“PHARMACOGNOSTICAL STUDIES & ANTI-INFLAMMATORY

EFFECT OF KAKAMACHI ON ALBINO RATS”.

AN EXPERIMENTAL STUDY BY

DDrr.. SShhiivvaakkuummaarr SS.. GGaannggoooorr..

Dissertation submitted to the Rajiv Gandhi University

of Health Sciences, Karnataka, Bangalore.

In partial fulfillment of Regulations for award of degree of

DDOOCCTTOORR OOFF MMEEDDIICCIINNEE ((AAYYUURRVVEEDDAA VVAACCHHAASSPPAATTII))

IN DRAVYAGUNA

Under the guidance of

DR. G. V. MULAGUND. M.D. (AYU)

PROFESSOR & H.O.D, DEPARTMENT OF

P. G. A. R. Center DRAVYAGUNA.

Under the co-guidance of

DR. KUBER. SANKH. M.D. (AYU)

LECTURER DEPARTMENT OF

P. G. A. R. Center DRAVYAGUNA.

POST GRADUATE DEPARTMENT OF DRAVYAGUNA D.G MELMALAGI AYURVEDIC MEDICAL COLLEGE AND RESEARCH

CENTER, GADAG - 582103.

2006

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.

DECLARATION BY THE CANDITATE

I hereby declare that this dissertation / thesis entitled

“PHARMACOGNOSTICAL STUDIES AND ANTI-INFLAMMATORY

EFFECT OF KAKAMACHI”- By an Experimental Study is a bonafide

and genuine research work carried out by me under the guidance of Dr. G. V.

MULAGUND. MD (Ayu),Professor and H.O.D, Post Graduate Department of

Dravyaguna, Shri D.G.M.A.M.C and Research center Gadag.

Date: Signature of the Candidate

Place: (Dr.Shivakumar. S. Gangoor)

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “PHARMACOGNOSTICAL

STUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ON

ALBINO RATS ” – BY an Experimental Study is a bonafide research work

done by Dr. Shivakumar. S. Gangoor in partial fulfillment of the requirement

for the degree of Ayurveda Vachaspati in Drvyaguna.

This work is applied, scientific and an original contribution in the field of

research in Ayurveda.

I am fully satisfied with his original work and recommended the dissertation to

be put before the adjudication.

Signature of the Guide

Dr. G. V. MULAGUND. M.D (AYU)

Professor and H.O.D

Post Graduate Department of

Dravyguna, D.G.M. Ayurvedic

Medical College,

Gadag.

ENDORSEMENT BY THE HOD, PRINCIPAL

HEAD OF THE INSTITUTION

This is to certify that the dissertation entitled “PHARMACOGNOSTICAL

STUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ON

ALBINO RATS ”- By an Experimental Study is a bonafide research work done by

Dr.Shivakumar S. Gangoor under the guidance of Dr. G. V.MULAGUND. MD

(Ayu) Professor and H.O.D, Post Graduate Department of Dravyaguna, Shri

D.G.M.A.M.C and research center Gadag and contributed good values to the

Ayurvedic research.

We here with forward this dissertation for the evaluation and adjudication.

Seal & Signature of the HOD Seal & Signature of the

Principal

Dr. G. V. Mulagund. Dr. G. B.Patil.

COPYRIGHT

Declaration by the Candidate

I here by declare that the Rajiv Gandhi University of Health Sciences,

Karnataka shall have the rights to preserve, use and disseminate this dissertation /

thesis in print or electronic format for academic / research purpose.

Date: Signature of the Candidate

Place: Dr.Shivakumar S. Gangoor.

© Rajiv Gandhi University of Health Sciences, Karnataka.

CERTIFICATE BY THE CO-GUIDE

This is to certify that the dissertation entitled “PHARMACOGNOSTICAL

STUDIES AND ANTI-INFLAMMATORY EFFECT OF KAKAMACHI ON

ALBINO RATS ” – BY an Experimental Study is a bonafide research work

done by Dr. Shivakumar. S. Gangoor in partial fulfillment of the requirement for

the degree of Ayurveda Vachaspati in Drvyaguna.

This work is applied, scientific and an original contribution in the field of research

in Ayurveda.

I am fully satisfied with his original work and recommended the dissertation to be

put before the adjudication.

Signature of the Co- Guide

Dr. KUBER.SANKH. M.D (AYU)

Lecturer,

Post Graduate Department of Dravyguna,

D.G.M. Ayurvedic Medical College,

Gadag.

ABSTRACT

Background :

Inflammation is fundamentally a protective response. These are exogenous

and endogenous stimuli can cause cell injury. Due to cell injury there will be

alterations in vascular caliber which leads to an increase in blood flow, changes in

micro vascular structure allow the plasma, proteins and leucocytes to leave the

circulation and get accumulated at the focus of injury leading to swelling. There are

multiple remedies available to reduce it with maximum side effects and

complications.

Inflammation is common pathological condition. It is livoited to any of the age

group nor to any socio-economic class of society. It is having greater influence of

treatment factors and conditions.

Since ancient times human societies have searched effective drugs of anti

inflammatory actions. Kakamachi is also a best example for an anti-inflammatory

drug.

The main objectives of the present protocol are

1) Pharmacognostical Study:

• Microscopic Evaluation

• Standardization and validation

• Extraction of phyto-chemical constituents.

• TLC, UV and I R.

2) Screening of fraction for anti-inflammatory activity.

3) To evaluate the anti-inflammatory effect of Kakamachi on Carreginine induced

albino rats in four different groups.

Method:

For thoroughly completion of present protocol needs fallowing essentialities such as

a) Source of data: present study

i) Literary source of data

ii) Experimental source of data

It includes phyto-chemical investigations of the extraction. i.e. fractionation

and characterization etc..

b) Method of Collection of Data:

In the present protocol the dried powder of Solanum nigrum extracted with

alcohol soxhlet apparatus is fractionated and is characterized. Fractionated principle

tested for anti-inflammatory activity by Carreginine induced Rat paw oedema.

a) Measurement by Carreginine induced inflammation in the hind paw of albino

rats.

b) Inflammation is measured before and after administration of Carreginine.

c) The Carreginine 0.01 ml is injected to the dorsum of the foot.

d) After one hour and three hours the readings were taken with the help of

plethismograph.

Observation;

In pharmacognostical study, morphologically colour of the leaves is green

with bitter taste. Shape is of ovate with characteristics acceptable odour and physical

evaluations of these respective entities are established. In phytochemical observations

of extractive and different preliminary phytochemical tests are Observed and

Identification by TLC are done and focused U V and I R spectroscopy observation on

the experimental studies are done on the basis of selection of Animal sample size,

preparation of the test drug.

Interpretations and Conclusions:

Different interoperations are done according to different points of view in

concern with the protocol and the study concern conclusions were drawn.

Key words

Kakamachi.

Solanum nigrum.

Pharmacognostical.

Experimental.

Anti-inflammatory.

ABBREVIATIONS

A.H. Ashtanga Hridaya

A.P.I Ayurvedic Pharmacopoeia of India

A.S Astang sangraha

B.P Bhava Prakashnighantu

C.S. Charaka Samhita

Chi. Chikitsasthana

D.N Dhanvantari Nighantu

Group C Control Group

Group S Standard Group

Group T Trial Group

K.N Kaideva Nighantu

M.N. Madanpala Nighantu

M.E.S. Maximal Electro-Shock

R.N. Raj Nighantu

S.N Sodal nighantu

S.S. Susharutha samhita

Sh. N Shaligram Nighantu

Su. Sutrasthana

T.L.C Thin Layer Chromatography

P.N. Priya Nighantu

Temp Temperature

D.W. Distilled water

ACKNOWLEDGEMENT

I express my deep sense of gratitude to the Principal Dr. G. B. Patil and

Management Committee for giving me the opportunity to pursue my post graduation

studies in this institution.

I am deeply inducted to my respected guide Dr. G. B. Mulgund M.D. (Ayu)

Prof. & HOD Dept. of P.G. Studies in Dravya Guna for his inspiring paramount

thoughts, zealous suggestions and guidance rendered for the successful completion of

this research work.

I also offer my deep felt gratitude to my co-guide Dr. Kuber Sankh M.D.

(Ayu) for his invaluable support without which the study could not have materialized.

I am thankful to associate guide Sri. B.S. Hunasi Lecturer Dept. of

Pharmacognosy B.L.D.E. Pahrmacy College, Bijapur. and Botanist Sri. S.A. Kapali

for their timely guidance and help. The completion of this dissertation has a lot to do

with their constant support.

I would like to express my profound gratitude to my department incharge Dr.

G.S. Hiremath M.D. (Ayu), Dr. Shashidhar Nidagundi and Dr. Smt. Veena Kori for

their guidance and inspiration given at various stages of my work.

I express my sincere thanks to Sri. Nandakumar for his help in statistical

analysis of results.

I am highly indebted to Dr. A.I. Akki, Dr. S.V. Teggi, Dr. V. M. Sabarar, Dr.

Wali, Dr. N.S. Shettar, Sri. Somashekahr T. for the able guidance and inspiration

given at different stages of my work.

I take this opportunity to thank the post graduate teaching staff of this

institution especially Dr. M.C. Patil, Dr. K.S.R. Prasad, Dr. Shivaramudu, Dr.

Shashidhar Dodamani and other lecturers of our college for their help and suggestions

during my post graduation studies.

I sincerely thank to my beloved classmates Dr. Jagadeesh H., Dr. Ashok

Bingi, Dr. Shivakumar Sajjan, Dr. Savita G., Dr. Anand, Dr. C.B. Inamdar and all

classmates of other post graduation branches for their constant co-operation and help.

I wish to convey my thanks to beloved librarian Sri. V.M. Mundinmani and

S.B. Sureban for providing essential references in the study.

I wish to convey my thanks to beloved friends Mr. S.M. Teggi, Mr. B.A.

Tenginkai, Mr. M.H. Vaijapur, Mr. R.M. Vaijapur for their co-operation.

I remember with respect my revered parents for their inspiration and

encouragement.

I sincerely thanks to Smt & Sri. S.B. Gangoor for their profound and never

ending love. I am grateful to my sisters Smt. Vijaylaxmi and Smt. Saraswati for their

affection. I am highly indebted to my mother-in-law and father-in-law Smt & Sri.

K.S. Anneppanavar for their affection and love.

This is incomplete without remembering my beloved wife Smt. Sunita who

helped in all respects to complete this valuable dissertation work and lastly son

Niranjan & lovely daughter Akshata for their affection.

I express my gratitude to all those who helped me directly or indirectly in

completing this work.

Date :

Place : Dr. S. S. Gangoor

CONTENTS

Sl. No Particulars Page No

i) Acknowledgement

ii) Abbreviations

iii) Abstract

iv) List of Tables

1. Introduction 1-3

2. Objectives 4

3. Review of literature 5-54

Drug Review

Disease Review

4. Methodology 55-70

Pharmacognostical study

Phytochemical study

Experimental study

5. Observations and Results 71-110

6. Discussion 111-119

7. Conclusion 120

8. Recommendation for further study 121

9. Summary 122-125

10. Bibliography

11. Annexure

Photographs

INTRODUCTION

INTRODUCTION

Ayurveda is a primordial system of medicine. The quest of the man is to live

happily, health is the elemental factor for happiness. The task of the medicine is to

preserve and restore the health by relieving the sufferings. Understanding the

medicine is essential to reach these both the goals. Pain is universally understood as a

sign of diseases. It is the most common symptom that brings a patient to physician's

attention.

The knowledge of this condition, to the modern medical science is just two

century old. This is well known to Ayurveda since 5000 years. The modern line of

treatment that provides a range of analgesic, physiotherapy and surgery but not a final

answer and there is a common problem of reoccurrence. An Ayurvedic approach is

helpful to improve the quality of life in-patient suffering from shotha.

Ayurvedic management measures seem to be more satisfactory because of

their simplicity, applicability, and easy availability and cost effectively. It is a serious

burning problem of the society. It is our contractual obligation to provide proper

management to the patients who are suffering from it and make them get through the

problem.

In the drug review, the different references from Samhitas, Vedas, in modern

books have been mentioned with different synonyms, having different important

valuable properties.

The useful part of Kakamachi has panchanga having with doses, churna 3 to 6

gms., kasaya 50 to 100 gms, patra swarasa 25 ml, phala churna 3 to 6 gms and uses

of Solanum nigrum according to external application and amayika prayoga.

The whole work comprises different sections. First section deals with the

Review of literature under various sub headings wherein literatures available from

1

INTRODUCTION

different references have been dealt. Material and Methods, Pharmacognostical study,

Phytochemical and Experimental studies are made use in this work. Observations and

Results, Discussions and Conclusion are given.

It is hoped that the humble efforts in the form of this dissertation will help in

understanding the effect of above mentioned measures as well as planning the future

research to find out the definite cure from Ayurvedic therapeutics.

Inflammation is well known by all the human beings because each person of

the world is certainly met with this condition at least once in their lifetime, which

affects all the age groups and to both the sexes. It makes the human beings to feel

fatigue, uneasiness, swelling, redness, and tenderness and disturb the day today life.

Shotha is well known to mankind since the beginning, its references are found

in Vedas. Which are the older written documents of knowledge, mentioned that

inflammation is first disease to incarnate on the earth. In all Samhita’s, the shotha is

explained in detail. "Charaka" who is well known for his skills of chikitsa explained.

It shows the redness, tenderness of the intolerable pain symptoms of shotha. Also

many others like Brihathrayees, Laghutrayees and recent authors were resolved

sufficient literature to explain the Shotha (Inflammation).

Inflammation is best defined in the teleogical terms, specifically it is a series

of molecules and cellular responses acquired during evaluation, designed to eliminate

foreign agents and promote repairs of damaged tissues. Unfortunately, these are not

infallible, pathogens have concurrently evolved inclusion to avoid elimination. New

pathogens occasionally emerge from the environment and under some circumstances

aberrant to inner inflammatory responses and damages the host. The complexity of

the inner inflammatory system is a reflection of millions of years of mental

challenges. It is becoming apparent that the system is not simple but rather a

2

INTRODUCTION

completely opposite of responses that were assembled approximately and where

elicited may synergize autogenic each other.

The cursory of inflammations are multitudinous. Almost anything that injures

living tissues there occurs the inflammation. By fast the most frequent causes of

inflammation is trauma, the minor injuries that escape our attention as well, and

infection by Bacteria, Viruses, Fungi as Parasites, to these must be added a host of

others cause exogenous and endogenous. Such as eradication, poisoning, metallic

disorders and derangements of the immune system. The body reacts to almost any

kind of injury by inflammation and other kinds of responses may follow but

inflammation is the first and most constant response.

In the present Phytochemical study of Solanum nigrum extraction, the

preliminary phytochemical tests were conducted and observed the presence or

absence of Proteins, Alkaloids, Saponnins, Tannins, Triterpenoids and Carbohydrates

with their characterizational findings, also with UV and IR methods and appropriate

techniques on identification by TLC method were done.

During experimental study total 24 albino Rats were taken and subjected them

in four groups (Group I to IV).

Group I was standard while Group II was trial group with minimum dose and

while group III was trial group with maximum dose and where as Group IV was

Vehicle group. Here Carreginine was induced to all the 24 Albino rats by injecting in

hind paw technique method. First inflammation on hind paw is observed and

measured. After induction of Carreginine to all the groups, immediately the

inflammation was observed and measured.

3

AIMS AND OBJECTIVES

AIMS AND OBJECTIVES

1. Pharmacognostical study-

• Pharmacological Evaluation.

• Microscopical Evaluation

• Standardization and Validation

• Extraction of Phyto chemical constituents

• T.L.C, UV IR

2. Screening of fraction for Anti-Inflammatory activity

3. To evaluate the Anti-Inflammatory effect of Kakamachi on Carageenen induced

albino rats in four different groups.

4

DRUG REVIEW

DRUG REVIEW

BOTANICAL NAME: Solanum nigrum.

i. KULA : KANTHAKARI.

II. GANAS

Charkokta ganas : Tikta skanda.

BOTANICAL CLASSIFICATION:

Classification according to Bentham and Hooker.

Table No : 1

Kingdom Plantae

Division Spermatophyta

Class Dicotyledonae

Sub-class Gamopetalae

Order Polemoniales

Series Bicarpellatae

Family Solanaceae

Genus Solanum

Species nigrum

According to Engler and Prantl.

Table No : 2

Kingdom Plantae

Division Embryophyta siphonogamy

Class Dicotyledon

Sub-class Sympetalae

Order Tubiflorae

Family Solanaceae

Genus Solanum

Species nigrum

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DRUG REVIEW

According to Hutchinson

Table No : 3

Phylum Angiospermae

Sub-phylum Dicotyledones

Division Herbaceae

Order Solanales

Family Solanaceae

Genus Solanum

Species nigrum

DESCRIPTION OF FAMILY SOLANACEAE

The family is spread over 90 genera and 2,000 species, Randle considered 85

genera and 2,000 species, according to others the number of species included under

this is as much as 2,200. The genus Solanum alone has about 1,700 species. In India

the family is represented by approximately 21 genera and about 70 species.

HABIT:

Majority of the plants are annual herbs, some are shrubs, rarely soft wood

trees. Some of the plants are climbing or a few armed with spines. The genus

Solanum with about 1,700 species shows diverse habits being herbs, shrubs, trees or

even creepers and woody climbers.

VEGETATIVE CHARACTERISTICS:

A branched taproot, the roots are thick and forked resembling human being in

miniature, stem erect, climbing, spinous, tuberous, herbaceous, sometimes woody,

branched, cylindrical, solid, sometimes hollow, hairy or glabrous. Leaves exstipulate,

6

DRUG REVIEW

petiolate, rarely sessile, usually alternate, sometimes opposite, simple entire,

sometimes pinnately divided, spinous, variegated, reticulate and unicostate.

FLORAL CHARACTERISTICS:

Inflorescence usually cymose, extra axillary and leaf opposed cymes, helicoid

cymes, axillary umbellate cymess, rarely helicoid, sometimes the flowers are solitary

or terminal in the main or lateral axis. Flowers bracteate or ebracteate, hypogynous,

bisexual, actinomorphic, complete sometimes zygomorphic, usually pentamerous,

variously coloured, large and showy in many species, sepals 5, fused 5, partite or

lobed –lobes sometimes 4-6, corolla tubular or companulate urceolate when mature,

ovate or imbricate, persistent, in Physalis calyx persistent and develops a bladder like

husk round the fruit, petals 5,fused,rotae, companulate, infundibular, lobed with 5 or

more lobes or bilabiate, ovate, sometimes imbricate, hairy on the outside variously

coloured, covered with stellate hairs. Stamens 5,sometimes 4, free, alternate with the

petals, epipetalous, filaments usually of unequal length, rarely the stamens are 4 and

are didinamous or only two, anthers two celled usually coming close together to form

a cone round the pistil, dehiscing by terminal pores or longitudinal slits, connective,

sometimes becoming tetra or tri to penta locular due to the formation of a false septa,

placentation axile, ovules many in each locule placed on a swollen and oblique

placenta. Fruit a berry, seeds small, numerous, smooth or pitted, endospermic,

endosperm fleshy, embryo curved.

FLORAL FORMULA

+ , K(5), C(5), A 5, G(2)

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DRUG REVIEW

FLORAL DIAGRAM

Figure No 1.

USEFUL PLANTS:

S. tuberosum, S. xanthocarpum, S. melongena, S. nigrum.

DESCRIPTION OF SOLANUM NIGRUM

COLLECTION:

Kakamachi patra is collected in varsharutu.

SYNONYMS

TABLE NO : 4

Bhavaprakash Nighantu Kakamachi,Dhwanksha, maachi, Kakahava, Vayasi.

Nighantu Adarsha Kakashva, Vayasi.

Dhanavantari Nighantu Kakhva, Vayasi, Katvi, Katuphala, Rasayanavara.

Raja Nighantu Vayasi, Kakamaachi, katu.

Kaideva Nighantu Kakasahava, kakamaachi, Kamata, Jaganephala,

Sarvatikta, Bahuphala, Swadupakaphala, Kamachi,

Kakini, Kushtaghni, Vayasi, Dhwanksha maachi,

Gudaphala, Rasayanavara, Katu.

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DRUG REVIEW

VERNACULAR NAMES

Arabic : Ambussalap, Enabeddir.

Assam : Pichakati

Bengal : Gurkamai, Kakmachi, Mako, Tulidun.

Bombay : Ghati, Kamuni, Mako.

English : Black Nightshade, Garden Nightshade, Hound's Berry, Morelle,

Petty morel.

Gujarat : Piludi.

Hindi : Gurukamai, Kabaiya, Makoi.

Latin : Solanum nigrum.

Malayalam : Trong parachichit.

Marathi : Ghati, Kakmachi, Kamoni, Laghukavali, Meko.

Persian : Rubahtareek

Portuguese : Herva moura, Solano.

Punjab : Kakmach, Kambei, Kwansaf, Mako, Riaungi.

Sanskrit : Bahuphala, Bahutikta, Dhvankshamachi, Ghanaghana,

Gucchaphala, Jaghenephala, Kaka, Kakamachi, Kakamata,

Kakini, Katuphala, Kushtaghni, Rasayani, Sarvatikta, Sundari,

Svadupaka, Tiktika, Vayasavha, Vayasi.

Sindha : Kanperum

Sinhalese : Kalukanweriya, Kalukungwareiya, Tibbatu.

Tamil : Manattakkali

Telugu : Gajuchettu, Kachi, Kakamachi, Kamachi, Kanchipundu, Kasaka.

Urdu : Makoya.

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DRUG REVIEW

TYPES:

In Nighantu two types are mentioned. They are

1. Shweta

2. Rakta

MORPHOLOGY OF SOLANUM NIGRUM

Solanum nigrum is wild herb growing for most part of the year in waste

places, sometimes reaching a height of 90 cm.

A variable annual, stem erect, glabrous or more less pubescent, much

divaricately branched. Leaves numerous, 2.5-9 by 2.5 cm, Ovate-lanceolate, subacute

or acuminate, thin, entire sinuate toothed, tapering in to the petiole; petioles 2 cm

long. Flowers small, in extra axillary subumbellate 3-8 flowered cymes; peduncles 6-

20 mm long, slender; pedicels 6-10 mm long, very slender. Calyx 3 mm long,

glabrous or nearly so; lobes 5, oblong, obtuse, 1.25 mm long, not enlarged in fruit.

Corolla 4-8 mm long, divided more than ½ way down into 5 oblong subacute lobes.

Filaments short, flattened, hairy at the base ; anthers 2.5 mm long, yellow, oblong,

obtuse, nothed at the apex. Ovary globose, glabrous; Style cylindric, hairy. Berry 6

mm in diameter, globose, usually purplish black but sometimes red or yellow, smooth

shining. Seeds discoid, 1.5 mm in diameter, minutely pitted, yellow. Calyx 5 sepals,

gamosepalous, lobes oblong, acute, white, vivate or imbricate aestivation.

Corolla- 5 petals, gamopetalous, rotate, lobes oblong, acute, white, twisted or ovate

aestivation.

Androecium- 5 stamens alternate to petals, polyandrous, epipetalous, filaments short,

anthers oblong, connivent in a cone like structures, bicelled, introse, yellow, dehiscing

by apical pores.

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DRUG REVIEW

Gynoecium- 2 carpals (bicarpellary), syncarpous, ovary obliquely placed in the

flower, ovary superior, globose, bilocular, axile placentation, many shining ovules

present on placenta, style simple hairy at the base, stigma one.

DISTRIBUTION:

Throughout India, Ceylon and all temperate and tropical regions of the world.

PROPERTIES

Table No : 5

Name of Text Rasa Guna Viryo Vipaka Karma Doshaghnata

Raja Nighantu Tika, Katu

Laghu Ushna Katu Vajeekara Rasayana Shotha hara

Tridosha hara

Dhanavantari Nighantu

Tikta Laghu Ushna Katu Swarya, Sarakam, Kushtanashaka, Vajeekara Rasayana

Tridosh hara

Kaideva Nighantu

Tikta Snigdha Ushna Katu Hridya

Nighantu Laghu

Shukra vardhaka, Swaraya, Saraka

Tridosha nashaka

Bhavaprakash Nighantu

Tikta, Katu

Snigdha, Ushna

Ushna Katu Swaraya, Shukra janana, Rasayana, Netraya, Shotha hara Kushtaghna

Tridosha nashaka

Priya Nighantu Tikta Ushna Katu Balya, Rasayana Shotha hara, Mutrala Yakritroga

Adarsha Nighantu

Katu Tikta

Ushna Katu Kushtaghna, Shothaghna Rasayana

Tridosha hara

Susruta Samhita Katu Tikta

Naatiushna Sheeta

Mushika visha nashaka, Tilla roga nasahaka

Tridosha hara

Charaka Samhita

Katu Tikta

Naatiushna Sheeta

Kushtaghnam Rasayanam

Tridosha hara

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DRUG REVIEW

PARTS USED

Pushpa, Phala, Panchaga, Beeja.

MATRA (DOSE) : Table No : 6

1. Swarasa 10-20 ml

2. Phala Choorna 1-3 gm

3. Arka 20-50 ml

TOXIC EFFECTS:

Excessive intake of fruit of Solanum nigrum leads to chardi, Trishna,

Udarashoola, Taraka Vispharana, Shirashool, Bhrama, Pralap, Akshepa, Sanyasa and

at the severity may lead to Death.

TREATMENT FOR TOXIC EFFECTS:

Treatment is as similar to Dhatura visha chikitsa.

USES OF SOLANUM NIGRUM

External Uses : Shotha hara, Vrunashodhana, Vedana sthapana, Savarnikarana.

Internal Uses :

Digestive System: Deepana, Yakrut uttejaka, Pittasaraka and Rechaka.

Circulatory System: Hridya, Rakta shodaka, Shothahara and reduces hypertension.

Respiratory System : Kaphaghna, Hikkanigrahana and Shwasahara.

Urinary System : Mootrala.

Skin : Swedajanana and Kushtaghna

Fever : Jwaraghna.

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DRUG REVIEW

THERAPEUTIC USES:

1. Useful in diseases of the heart , eyes, piles, leucoderma, itch, worms in the ear,

dysentary, hiccough, vomitting, asthama, bronchitis, fever, urinary discharges,

improves the voice, favour conception and facilitate delivary and rat bite.

2. The root bark is laxative and useful in the diseases of the ear, eye, and the

nose. Good for ulcers on the neck, burning of the throat, inflammation of the

liver, chronic fever, gripping and is harmful to the pregnant women.

3. The Leaves have a bad odour and taste and used for headache and the diseases

of the nose.

4. The fruit is useful in thirst due to fever and inflammation.

5. The seeds are laxative and are useful in giddiness, gonorrhea, thirst and

inflammation.

6. In Bengal the berrys are employed in fever, diarrhoea, eye diseases and

hydrophobia.

7. The juice in doses of six to eight ounces is given in the treatment of chronic

enlargement of the liver and considered as valuable alternative. It acts as a

hydrogogue catharic and diuretic.

8. The syrup acts as an expectorant and diaphoretic, used as a cooling drink in

fevers.

9. In North-western province the juice is used in blood spitting, piles, dysentary

etc.

10. A decoction of the leaves in the dosage of drachm thrice daily in the treatment

of dropsical affectin by the mooden sheriff. Its action is diuretic and laxative.

11. The young shoots are given in the chronic skin diseases and psoriasis in the

konkan region.

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12. The Chinese employ the juice of the leaves to alleviate the pain in

inflammation of the kidneys , bladder and in virulent in gonorrhoea.

13. The plant is given internally for cardialgia and externally in nephritic colic,

corroding ulcer, suppurating chancre and in severe burnings and herpes.

14. The decoction of the leaves is used as diuretic and depurative.

15. The plant is used by Europians for convulsions. The herb is one of the native

remedies for local application to anthrax pustules and a paste of the green

berries is applied to ringworm. The Xosas use the plant for disinfecting

anthrax infected meat.

16. The Zulu's administered as an enema to infants with abdominal upsets. The

Suto's rub the burnt and powdered root in to the incisions on the back for relief

of lumbgo.

17. The fruit in combination with other drug is prescribed for snake bite and

scorpion sting, but it is not an antidote to either snake venom or scorpion

venom.

18. The leaves are employed as poultice over rheumatic and gouty joints and as a

remedy in skin diseases.

19. Freshly prepared fluid extract from all the portions of the plant has been

recommended in dropsy, in heart diseases, skin diseases, piles, gonorrhoea,

inflammatory swellings and chronic cirrhosis of the liver and seen in doses of

½ to 2 drachms.

20. A Syrup of the plant is useful as an cooling drink in fevers and promotes

perspiration.

21. Heated warm leaves are applied to the painful and swollen part of the testicles.

22. Decoction of the berries and flowers are useful in cough.

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viii) CHEMICAL COMPOSITION

The analysis of the leaves gave the following values in 100 gm of edible

material.

Moisture- 82.1

Protein- 5.9

Fat- 1.0

Minerals- 2.1

Carbohydrates- 8.9

Calcium- 410 mg

Phosphorus- 70 mg

Iron- 20.5

Leaf is a rich source of Riboflavin. The values for various vitamins present in the leaf

are (in 100 mg edible material)

Riboflavin- 0.59

Nicotanic acid- 0.92

Vitamin C- 11 mg

The higher values for the vitamin C (20-40 mg) have been reported.

The fruits contain-

Glucose-15-20%

Vitamin –C

B-Carotene

Green unripe fruits, however contain glycolalkaloids, consumption of the

same leads to toxic hazards to human beings as well as to the livestocks. Ripe fruit

contains very little alkaloids and can be consumed without ill effects.

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Seeds forming 9.5 percent of protein on dry weight basis. They yield a

greenish yellow oil-21.5 % with the following physical and chemical constituents-

Specific gravity-0.9198

Acid value- 11.62

Saponification value- 184.0

Acet value- 25.7

Iod value- 123.2

Hehner value- 92.9

R M value- 0.66

Unsapon matter- 1.4 %

The component fatty acids of the oil are

Linolic- 46.63

Oleic- 49.73

Palmitic- 1.76

Steraic- 1.88

The unsaponifiable matter contains sitosterol.

Immature green fruit of the plant contains four steroidal glycol alkaloids viz;

• Solamargine

• Solasonine

• Solanigrine

• Solanigrinine

All these yeild Solasodine as the aglycone. It also contains a steroidal genin,

tigogenin. (0.101 %) Solanigrine and (0.431 %) Solasonine are present in leaves

respectively.

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ECONOMIC IMPORTANCE :

Economically the Solanaceae family is fairly important. It comprises several

plants of food value, medicinal value, vegetable and ornamental values. In which

Solanum nigrum posses medicinal properties. The fruits are given in fevers, diarrhoea,

eye diseases and hydrophobia. The juice of plant is given in chronic enlargements of

the liver, in bleeding piles, dysentery. The fruits are edible and very much liked by the

children.

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REVIEW OF INFLAMMATION

1. INTRODUCTION

There are many features in the normal structure and physiologies of the body,

which serve, protect it against injury. The continuity of skin and mucous surfaces

protects to a considerable extent against minor injuries. Normal or increased secretion

from nose, eyes, urinary tract. Movements like that of eyelids or cilia of the tracheal

mucosa chemical constituents such as of gastric juice is other instances of natural

defense mechanism against injury or bacterial invasion. Reflexes such as sneezing and

cough are definite protective mechanisms, immune bodies present in the body fluids,

which may be increased by the survival of natural disease and by the artificial

processes, endow the organism with an important means of defense.

Inflammation in addition to above means seems to be a better defensive

reaction against an injury locally. It may be associated with general changes such as

fires, leukocytosis and development of immune substances, but it itself operates only

at the site of injury.

The same exogenous and endogenous stimuli that causes cell injury also elicit

a complex reaction in vascularized connective tissues called inflammation. Reduced

to its simplest terms, inflammation is a protective response intended to eliminate the

initial cause of cell injury as well as the necrotic cells and tissues resulting from the

original insult. Inflammation accomplishes its protective mission by dilating,

destroying or otherwise neutralizing harmful agonists (e.g. microbes or toxins). It thus

sets into motion the events that eventually heal and reconstitute the sites of injury.

Thus inflammation is also intimately interviews with repair processes whereby

damaged tissue is replaced by the regeneration of parenchymal cells, and loss by

filling of any residual defect fibrous scar tissues.

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Although inflammation helps to clear infections and repairs, makes wound

healing possible, both inflammation and repairs have considerable potential to cause

harm. Thus inflammatory responses are the basis of life threatening anaphylatic

reactions to insect bites or drugs, as well as to cause certain diseases such as

rheumatoid arthritis and athero-sclerosis, other harmful examples include

inflammation in the peritoneum leading to fibrous bands that cause intestinal

obstruction or pericardial inflammation resulting in lenses encasing scar that impairs

cardiac function.

The inflammatory response has many roles; these include circulating cells and

plasma proteins, vascular wall cells and extra cellular matrix of the surrounding

connective tissue. The circulating cells include bone marrow derived polymorpho-

nucleus. Leukocytes (neutrophils), eosinophils, basophils, lymphocytes, monocytes

and platelets, the circulating proteins include clotting factors; kininogens and

complement components, largely synthesized by the vascular wall cells include the

endothelial cells in direct contact with the blood as well as the underlying smooth

muscle cells that are important to the vessels. The connective tissue cells include

sentinels, macrophages and lymphocytes, in addition to the fibroblasts that synthesize

the extra cellular matrix and can proliferate to fill in a wound, the extra cellular matrix

(ECM) consist of fibrous structural proteins (e.g. collagen and elastin), get forming

proteglycous and adhesive glycoprotein (e.g. fibronectin) that are the cells – FCM

=ECM-ECM connectors, as we see, these all interact to resolve local injury and

restore normal tissue function.

Inflammation is divided into two basic patterns. Acute inflammation is of

relatively short duration, lasting from a few minutes to a few days and is characterized

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by fluid and plasma protein exudation, a predominantly neutrophilic leukocyte

accumulation. Chronic inflammation is of longer duration (day to years) and typical

by influx of lymphocytes and macrophages associated with vascular proliferation and

scarring. However these basic forms of inflammation may have many factors and

histological appearances.

The reaction of vascularized living tissue to local injury, invertebrates with no

vascular system, single celled organisms, and multicellular parasites have their own

responses to local injury. These include phagocytosis of the injurious agents,

endropment of the irritants by specialized cells haemocytes, which ingest it and

neutralization of noxious stimuli by hypertrophy of the cell or one of its organelles.

The inflammatory process in higher forms is the reaction of blood vessels leading to

the accumulation of fluid and blood cells.

The inflammatory response is closely interwined with the process of repair, it

serves to destroy, dilute the injurious agent, but sets into motion a complex series of

events, heal and reconstitute the damaged tissue as far as possible.

2. DEFINITION AND TERMINOLOGY

DEFINITION:

Inflammation is defined as the local response of living mammalian tissues to

injury due to any agent. It is a body defense reaction in order to eliminate or limit the

spread of injurious agent as well as to remove the consequent necrosed cells and

tissues.

TERMINOLOGY:

The majority of inflammatory serous are named by adding the suffix It is to

the anglicized form of the Greek hour of the tissue or organ involved. Thus we speak

of dermatitis, gastritis, meningitis and so on.

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The suffix It is derived from the Greek for belonging to the names ending in It

is do not imply the cause of the inflammation. For example, dermatitis may be due to

the infection or chemical injury, radiation or immunologic reaction and so on. All the

name implies is inflammation of the skin.

Inflammatory diseases have been known since ancient times and like others of

ancient lineage, they sometimes bear strange names derived from the past Erysipelas a

spreading Streptococcal infection of the subcutaneous tissue of the face was named by

the great Hippocrates 300 years before christ pneumonia is another old term still in

common use, these terms will be defined when we come to the diseases of the organs

affected.

Inflammation is best defined in teleological terms, specifically it is a series of

molecules and cellular responses acquired during evaluation designed to eliminate

foreign agents and promote repair of damaged tissues. Unfortunately, these responses

are not infallible. Pathogens have concurrently evolved mechanism to avoid

elimination; new pathogens occasionally emerge from the environment and under

some circumstances aberrant immuno inflammatory response damages the host. The

complexity of the immuno inflammatory system is a reflection of millions of years of

environmental challenges. It is becoming apparent that the system is not simple but

rather a complex composite of responses that were assembled opportunitically and

when elicited may synergize or antagonize each other.

3. HISTORICAL BACKGROUND

For centuries, humans have identified inflammation fire, undoubtedly, as a

result of the experience of reduces heat and pain associated its occurrence.

Interestingly scientific investigation of inflammation has extended this analogy. At

the microscopic level, inflammation is described as an accumulation of leukocytes

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that “spread” within tissues and then ultimately “burn out” and heal or lead

“smoldering’ conditions. Similarly at the molecular level, leukocytes use an oxidative

mechanism in essence a form of biologic fire that destroys micro-organisms and

damages tissues. Despite the essential truth of our intuitive sense of inflammation, the

objective under starting has come slowly.

At least since the eighteenth century, inflammation has been considered as

manifestation of immunity, but the mechanisms governing inflammatory events have

remained enigmatic until recent decades. During the late nineteenth and much of the

twentieth centuries, important advances were made regard to manipulating gum oral

immunity as evidenced by the development of vaccines and the use of antiserum.

These great advances tended to over shadow the role of phagocyte cells in resistance

to infection as promoted by the Zoologist Elie Metchnikoff like wise

histopathologists. Such as Rudolf Virechow and Julius Cohnheim speculated that

cellulus and vascular components of inflammation were critical elements, but they

could not ascertain full significance or relationship to humeral immunity. It is now

understood that inflammation is a dynamic interaction of humeral of cellular

responses. Recently studies of inflammation have focused on revealing the

“molecules language” that dictates the observed events clearly. There is a complex era

of both humeral and cellular signals that determine the quality, intensity and duration

of inflammation.

The features of inflammation were described in an Egyptian paper around

3000 BC. celsus, a Roman writer of the first century AD listed the four cardinal signs

of inflammation, they were rubor (redness) tumor (swelling), calor (heat) and dolor

(pain). These signs are more prominent in acute inflammation than in chronic

inflammation. Later Virechow added the fifth sign of functiolaesa (loss of function).

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Inflammation is not a disease but a non-specific response that has a saluton

effect on its host, this obvious fact was noticed by the Scottish surgeon John Hunter in

1793.

Julius Cohnheim (1839-1884) who provided one of the first and best

microscopic description of inflammation and observed inflamed blood vessels in thin

and transparent membranes such as in the mesentery and tongue of frog. Noting the

initial vasolidation and changes in blood flow, the subsequent oedema caused by

increased vascular permeability and the characteristic leukocyte emigration.

Elie Metchnikoff, a Russian biologist in 1882 discovered the process of

phagocytosis by observing the ingestion of rose thorns by amebocytes of starfish

larvae and of bacteria by mammalian leukocytes and concluded that the purpose of

inflammation was to bring phagocytic cells to the injured area to engulf invading

bacteria. Metchnikoff contradicted the prevailing theory, that the purpose of

inflammation (humoral theory of immunity) was to bring in factors from the serum to

neutrilize the infectious agents. It became clear that both cells (phagocytes) and serum

factors (antibodies) were critical for defense against micro-organisms, this

strengthened by the discovery of the antitoxins by Behring and Kitasato (1890)

On the basis of simple experimental studies of inflammatory response on skin.

Sir Thomas Lewis established the concept that chemical substances such as histamine

locally induced by injury, mediate the vascular changes of inflammation, this

fundamental concept underlies the important discoveries of chemical mediators of

inflammation and the use of anti-inflammatory agents.

4. CAUSES OF INFLAMMATION

1. The outstanding cause is injury by living agents; these can induce progressive

inflammation due to two basic procones, early inflammatory response and

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healing. These processes generally have protective role against injurious

agents. Inflammation is distinct from infection, is being a protective response

by the body and invasion into the body by harmful microbes and their ill

effects by toxins. The causes may be due to physical agents i.e., heat, cold

radiation, mechanical trauma.

2. Chemical agents i.e., organic and inorganic poisons

3. Infective agents like bacteria, viruses and their toxins

4. Immunological agents like cell mediated and antigen antibody reactions.

CAUSES OF INFLAMMATION

CHART NO – 1

INJURIOUS AGENTS

Living agents Non-living agents

Physical agents Chemical agents

I) Mechanical trauma I) Strong acids

ii) Presence of foreign body ii) Strong alkalies

iii) Under heat and cold iii) poisons

iv) Pressure

v) Ionising radiation

5. SIGNS AND SYMPTOMS

The Roman writer Celsus described four cardinal signs of inflammation as

(a) Rubor (Redness): An acutely inflamed tissue appears red due to dilatation of

small blood vessels within the damaged area.

E.g., Skin affected by sunburn, cellulites by bacterial infection etc.

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(b) Calor (Heat): Increase in temperature is seen only in peripheral parts of the

body such as skin. It is due to increased blood flow (hyperaemia) through the

region resulting in vascular dilatation and delivery of warm blood to the area.

E.g., Systemic fever.

(c) Tumor (Swelling): Swelling results from oedema, the accumulation of fluid in

the extra vascular space as part of the fluid exudates and to a much lesser

extent, from the physical mass of the inflammatory cells migrating into the

area.

(d) Dolor (Pain): Pain is the best-known feature of acute inflammation. It results

partly from stretching and distortion of tissues due to inflammatory oedema

and in particular from pus under pressure in an abscess cavity. Chemical

mediators like bradykinin, prostglandins and serotonin are known to induce

pain.

(e) Loss of function (Functio laesa): Movement of an inflamed area is consciously

and reflexly inhibited by pain, while severe swelling may physically

immobilize the tissues.

6. CLASSIFICATION OF INFLAMMATION

CHART NO-2

CLASSIFICATION OF INFLAMMATION

Acute inflammation Sub Acute inflammation Chronic inflammation

i) Catarrhal 1) Diffused

ii) Fibrinous or Serofibrinous 2) Suppurative

iii) Suppurative or purulent 3) Granulomatous

iv) Haemorrhagic 4) Fibrinoid

v) Pseudo membranous

vi) Allergic

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I. ACUTE INFLAMMATION:

a) CATARRHAL ACUTE INFLAMMATION:

This is confined to mucous membrane, the mucous cells of the lining and of

the mucous glands secrete large quantities of mucin, there is molecular desquamation

of the surface lining, not so pronounced as to appear as ulceration. The discharge on

the surface or in the lumen consists of mucous plasma, leucocytes and desquamated

epithelial cells, it is mucoid in the early stages and becomes mucopurulent later.

Acute catarrhal inflammation is mildest form of inflammation, but in some organs it

can progress to termination and can become chronic. E.g. Rhinitis of common cold,

catarrhal bronchitis.

b) FIBRINOUS OR SEROFIBRINOUS INFLAMMATION:

This is seen in the serous membranes, the pericardium, pleura, peritoneum etc.

It consists of plenty of fibrin in the exudates derived from the exudate plasma,

irrespective of the nature of the irritant, they react in a stereotyped manner.

Neutrophils and macrophages are seen in the fibrinous network.

c) SUPPURATIVE OR PURULENT INFLAMMATION:

This type is characterized by the formation of pus. In solid tissues and organs

suppurative inflammation causes Abscesses, in contrast to the circumscribed affection

in abscess formation the process may become diffuse and spreading. Diffuse lesions

in the connective tissue are described as “Phlegmonous Inflammation or Cellulites”.

d) ACUTE HAEMORRHAGIC INFLAMMATION:

In certain cases, bacterial toxins produce intense injury to the capillary wall

and allow large number of Red Blood Corpuscles to escape, making the exudates

haemorrhagic. E.g. In Influenza Pneumonia, Acute Glomerulonephritis etc.

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e) PSEUDO MEMBRANOUS INFLAMMATION:

This form of inflammatory reaction is characterized by the formation of a

membrane usually made up of precipitated fibrin, necrotic epithelium and

inflammatory white cells. The reaction is encountered only on mucosal surfaces

commonly in the Pharynx, Larynx, Respiratory passage etc. The membrane formation

results from an acute inflammation response to a powerful necrotizing toxin, out

pouring of exudates traps the necrotic and cellular debris producing a dirty gray-

white, rubbery membrane on the eroded surfaces. E.g. In Diphtheria.

f) ALLERGIC INFLAMMATION:

In this, Inflammation is primarily and exclusively by antigen-antibody

reactions. The injury in this is due to the occurrence of antigen-antibody union on

walls of the tissue cells, hypersensitivity has much greater significance. The vascular

changes are associated with a cellular exudates consisting of neutrophils, eosinophils,

plasma cells and macrophages. Eosinophils may be spectacular components, tendency

to necrosis and tissue decay due to accelerated reaction and increase in the Phagocytic

power of the Leucocytes with increase of eosinophils are distinct features in allergic

inflammation.

2. CHRONIC INFLAMMATION

The acute inflammatory response is unable to remove or neutralize an

injurious agent; the response is modified to chronic. It is not usual for a chronic

response to last many months but years. As the inflammatory process continues, fluid

exudates diminishes and cellular response assumes dominance, the chronic response

is dominated by a massive build up of cells in the affected tissue. These cells are

primarily macrophages and lymphocytes. In chronic inflammation the agent and the

host are just capable of resisting each other. The agents involved are of low inevitable

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ability. They are unable to penetrate deeply in to or spread throughout the body of the

host. Such agents may be bacteria, fungi, larger parasites. Foreign bodies which are

insoluble in body’s fluid can also elicit a chronic inflammatory response.

Regardless of the specific nature of the inciting agent, its presence in the tissue

promotes a long term conflict with the phagocytic cells of the host; heavy infiltration

by the inflammatory cells progressively interferes with normal function. When the

process continues over a month and years, the function detoriates as tissue is

destroyed, accumulating inflammatory cells replace functional tissues and scarring

develops. This deterioration ultimately leads to somatic death.

TYPES OF CHRONIC INFLAMMATIONS

Following Four types are included in Chronic Inflammation:

1) Chronic Diffused Inflammation

2) Chronic Suppurative Inflammation

3) Chronic Granulomatous Inflammation and

4) Chronic Fibrinoid Inflammation.

1) CHRONIC DIFFUSED INFLAMMATION:

Exudates, which may be diffused or focal shows lymphocytes, plasma cells

and macrophages, under certain stimuli macrophages develop into epitheloid cells and

multinucleated giant cells. Fibroblasts are present, in older lesions fibrosis

conspicuous. E.g. Chronic Ulcer.

2) CHRONIC SUPPURATIVE INFLAMMATION:

It is a non-specific inflammatory cell infilteration, in which infiltration by

polymorphs and abscess formation. E.g. Actinomycosis

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3) CHRONIC GRANULOMATOUS INFLAMMATION:

It is characterized by the formation of Granulomas, a tiny lesion composed of

epithelial cells and lymphoid cells at the periphery, also granulomas may have giant

cells, necrosis and fibrosis. It is seen in specific infective granulomas as in

Tuberculosis, syphilis. Leprosy etc.

4) CHRONIC FIBRINOID INFLAMMATION:

It is a degenerative phenomenon like rheumatoid arthritis, reheumatic fever

etc.

DIFFERENCES BETWEEN ACUTE INFLAMMATION & CHRONIC

INFLAMMATION

Table No 7

Acute inflammation Chronic Inflammation

1.Duration: Usually for days or weeks 1. Duration: For months or Years

2. Cardinal Signs: Present 2. Cardinal Signs: Doubtful or not

perceptible

3.Vascular Changes: Present 3.Vascular Changes: Not Marked

4. Exudation of Plasma: Present 4. Exudation of Plasma: Doubtful or

Absent

5. Cellular Exudates:

Neutrophils initially lates Macrophages and

Fibroblasts Stage of repair Lymphocytes

are few.

5.Cellular Exudates:

Histocytes Plasma cells lymphocytes

Fibroblasts are present Neutrophils

absent or very less in numbers.

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7. GENERAL MECHANISM OF INFLAMMATION

The starting point of inflammation is the cell damage, living or inert, the cells

which do not damage are not capable of producing inflammation. However once the

damage has occurred the reaction takes place inevitably and proceeds through a

definite series of events to its ultimate end, i.e. repair of damage and restoration of

function.

CHART NO- 3

GENERAL MECHANISM OF INFLAMMATION

• Vascular phenomenon changes

Vasodilation

Active hyperemia

Capillary dilation

Static of blood

Sludging of red cells in the capillaries

Pavementation of leukocytes

Exudation of fluid and out pouring of polymorphs

• Cellular response

Local

Exudation

Fluid of exudation

Cells of the exudation

General response

Fever degeneration

Leukocytosis

• Repair of tissues

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Degenerative

Albuminous degeneration

a. Repuls degeneration

b. Healing cloudy swelling

c. Regeneration

Fatty degeneration

a. Suppuration

b. Abusers

c. Boil

d. Carbuncle

e. Cellulites

Proliferative

Phagocytes

a. Attachment

b. Engulfment

(i) Azophil

(ii) Special groused

c. Killing and degeneration

(i) Oxygen dependent mechanism

(ii) Oxygen independent mechanism

Cells included in the inflammatory exudates are :

• Neutrophil

• Eosinophils

• Mast cells

• Lymphocytes

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• Plasma cells

• Macrophages

MECHANISM OF INFLAMMATION -

Management of inflammation and action of non-steroidal anti-inflammatory drugs

(NSAID)

Irrespective of the nature of causative factors or the type of inflammation, the

tissue reaction in first few hours is stereotyped and similar. Its pathology can be

understood in the fallowing steps.

1.Vascular phenomenon

2.Cellular phenomenon

3.Repair

1.Vascular phenomenon:

Following changes will occur in vascular phenomenon one by one

i. Vasodilatation: Proceeded by transient vasoconstriction

ii. Active hyperemia

iii. Capillary dilatation

iv. Stasis of Blood

v. Sludging of red cells in the capillaries.

vi. Pavementation of leukocytes.

vii. Exudation of fluid and out pouring of polymorphs.

There are number of cells present in the inflammatory exudates which perform

various functions. Those are

a. Neutrophils

b. Eosinophils

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c. Mast cells

d. Lymphocytes

e. Plasma cells

f. Macrophages

A brief description and the role is as follows.

a) Neutrophils: Neutrophils or Polymorphs are the cells along with Basophils and

Eosinophils are known as granulocytes, due to the presence of granules in the

cytoplasm, contains substances like proteases,myloperoxidase and alkaline

phosphates. Neutrophils are actively motile, get collected arround blood

vessels and passes through the tissue by active amoeboid movements, there

from the first line of defense in bacterial infections.

b) Eosinophils: These are larger than the Neutrophils and constitute 2-4% of the

total blood leucocytes. They appear only at the sites of inflammatory exudates

of diseases of immunological origin. They remain in the circulation for a short

peroid and rapidly attracted in the tiisues by the raised concentration of

released histamine. Eosinophils are phagocytic in nature. These may share

many structural and functional similarities with neutrophils, like their

production in the bone marrow, locomotion, lobed nucleus and presence of

granules in the cytoplasm. Granules of eosinophils are rich in

myeloperoxidase than neutrophils and lack lysozyme. High level of steroid

harmone (eosinopenia) leads to fall in number, and even disappear from the

blood. These may be increased in certain conditions like Allergy, Parasitic

infestations, skin diseases and certain malignant lymphomas.

c) Mast cells: These cells contain coarse basophilic granules in the cytoplasm

and a polymorphous nucleus, These granules are laden with heparin and

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histamine. The functions of mast cells in normal Human being is not vivid.

They are believed to produce the acid.

d) Plasma cells: These are larger than the lymphocytes with more abundant

cytoplasm and an eccentric nucleus. These cells are normally not seen in

peripheral blood. They develop from lymphocytes and are rich in RNA and

Gama-globulin in their cytoplasm. There is an inter relationship between

Plasmocytosis and Hyperglobulinaemia. These cells are most active in

antibody synthesis. The number will increase when prolonged infection with

immunological responses. e.g. Syphilis, Rheumatoid Arthritis, Tuberculosis,

Hypersensitivity states and multiple myeloma.

e) Macrophages: These are large mono-nucleated cells play an important role in

the stages of Acute and Chronic Inflammations. It is believed that many

lymphocytes derived from the blood are converted in to microphages at the

site of inflammation, by increase in cytoplasm and enlargement of the nucleus

and are derived from Kuffer cells of the liver and histocytes. They form the

scavenger cells of inflammation, combine to form giant cells and need

phagocytic action. The giant cells are mainly of :

i. Tumour Giant Cells

a. Anaplastic Cancer Giant Cells

b. Reed-Sternberg Giant Cells and

c. Giant Cells of Tumour

ii. Foreign Body Giant Cells.

i. Tumour Giant Cells: It is of fallowing types

a. Anaplastic Cancer Giant Cells:

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These are larger and have numerous nuclei which are hyper chromatic and

vary in size. These giant cells are not derived from macrophages but are formed from

dividing nuclei of the neoplastic cells.

b. Reed-Sternberg Giant Cells:

These are malignant tumour giant cells , which are binucleate and are seen in

various histologic types of Hodgkin's lymphomas.

c. Giant Cells of Tumour:

These cells are usually found in the bones, uniform distribution and

osteoclastic giant cells spread in the stroma.

ii. Foreign Body Giant Cells:-

These contain numerous nuclei, which are uniform in size and shape and

resemble the nuclei of macrophages, these nuclei are scattered throughout the

cytoplasm, these are usually seen in the chronic infective granulomas.

2.CELLULAR RESPONSE:

Cellular response can be explained in to a) Local response and b) General

response. The local response starts with the pavementing and emigration of

leukocytes before the cells of the damaged tissue release some chemicals that bring

about all the changes. The Vasodilation and increased permeability is due to the

substances like "leukotoxin" and "histamines" are produced by the damaged tissue

cells.

These stimuli activate both Hematogenic and Histogenic cells, which carry out

various functions like vascular phenomenon, pavementation and emigration of

leukocytes and various other cellular responses.

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Following a local injury, there is a transient constriction in the vessels that

cause local Ischemia. This mommentary constriction is followed by an active phase of

hyperemia with dilation of vessels, this dilation chiefly occurs in arteries and venules

and at last in the capillary bed. The active hyperemia lasts for a few hours. The

capillaries get changed and become prominent.

The blood vessels keep on dilating and the active hyperemia is fallowed by the

stagnation of blood, which is termed as stasis and the clotting takes place in the

vessels. The blood supply to the tissue is lost and necrosis sets in the vasodilation that

follows the transient vasoconstriction (due to neural reflex) It is recognized that a

sensory fibre has a vasodilator branch to an arteriole. When there is an injury, there is

a reflex vasoconstriction and thus vasodilation sets in.

The vasodilation occurs under the influence of chemical mediators also, it was

postulated by Lewis (1927) that "H" like substance cause vasodilation. Further

Menklin postulated about "Leukotoxin". In addition, it increases the vascular

permeability and responsible for the emigration of leukocytes, the slowing of blood

flow in the vessels may be attributed to swelling of the endothelial lining of the blood

vessels, vasodilatation that decreases the pressure. Loss of blood fluid in interstitial

spaces makes the blood viscous. There is sludging of red cells. The red cells become

sticky and adhere to one another in masses and to the walls of the vessels.

In this slower blood stream, rearrangement of the corpuscles takes place, under

normal condition, the red and white cells flow intermingled in the central part of the

vessels forming an axial stream, which is separated from the wall by a clear plasmatic

zone free from cells. When there is some degree of injury, the leukocytes fall out of

the axial stream and come to occupy the plasmatic zone adhere to the vessel wall and

seem to drag themselves along with difficulty. In this way, the inner wall of the

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DISEASE REVIEW

capillary becomes paved by a broken line of leukocytes without the admixture of a

single red blood cell. This arrangement is called pavementing of leukocytes.

Normally the endothelial cells of the blood vessels and blood cells repel one

another due to change in the electrical potential, where these carry negative charges

with them, hence the repulsion. The endothelium becomes positvely charged and the

leukocytes are the first of the cellular elements to be attracted to and adhere to the

lining of the vessel.

The endothelial cells also become enlarged, proliferated and thus assume

round shape. The inter-endothelial space widens and through these spaces leukocytes

emigrate.

A) EXUDATION:

After dilation of blood vessels, the solid and fluid contents of plasma as well

as of the blood cells pass through the vessel walls and constitute within the tissues.

Thus, the fluid is rich in protein contents and the cells constitute the exudates.

B) FLUID OF THE EXUDATE:

Normally the walls of the blood vessels are permeable to the fluid, some ionic

salts and the molecules with molecular weight less than 10,000 Daltons, large part of

the fluid get reabsorbed and the remaining is carried by lymph channels.

But during inflammation, this exudate fluid, which originates from plasma

differs from it in several aspects. Its solid contents are almost double than the contents

of the lymph. Its specific gravity is 1.020. The main reason is that serum albumin and

serum globulin is present in exudate. The fluid molecules come out of vessels due to

increased permeability of vessels.

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DISEASE REVIEW

Besides this protein, the exudate contains various extractions like Urea, fibrin

forming elements, mucin ferments and immune bodies, oxidases, lipases and trysin.

All type of immune bodies may be found including cytolysin, haemolysin,

bacteriolysin, agglutinins, opsonins and complement fixing bodies. The formats and

the immune substances are distinctly higher in oedema than oedema produced due to

other causes.

FUNCTION OF FLUIDS OF EXUDATE :

1. It dilutes the soluble poisons and irritates, thereby reduces their direct effect.

2. It provides over runs of escape for the metabolites which are formed in excess.

3. It maintains normal hydrogen concentration.

The protolytic enzymes serve to complete the solution of the tissues which

have been injured or killed, thus aid in their removal. Exudate is rich in fibrin forming

proteins. The environment in which it is present favours fibrin formation, this serves

as the limits of extent of the inflammatory process. The fibrin mesh in the lymphatics

serve as a filter and with solid materials especially bacteria, if it is not dissolving by

protolytic enzymes it provides a frame work on which connective tissue grows and

healing takes place.

C) CELLS OF THE EXUDATES:

After pavementing the leukocytes emigrate in extra vascular spaces, as they

emerge from the outer margin of endothelium, a new basement membrane forms

between them and the endothelium disappears permitting release of the leukocytes in

to the extra vascular space without leaving any defect behind them, under the

influence of various chemical mediators, this phenomenon is known as "Chemotaxis".

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DISEASE REVIEW

D) PHAGOCYTES:

Phagocytes can be resolved in to three distinct ways

1. Attachment of the Particle to the surface of the phagocyte.

2. Engulfment.

3. Killing and degeneration of the ingested microbe or particle.

1. Attachment of the Particle to the surface of the phagocyte:

These cells are recognized and attracted to bacteria by chemotactic factors

released by bacterial products as well as by tissue proteins, in order to establish a

bond between bacteria and the cell membrane of phagocytic cells, the micro

organisms get coated with opsonins which are naturally occurring factors in the

serum.

The main opsonin present in the serum IgG opsonin, is the Fe fragment of

immunoglobulin G, antibody in the serum that coats the bacteria. C3b opsonin is the

fragment of completement, which is generated by activation of completement

pathway.

Lactins are carbohydrate-bonding proteins in the plasma, which bind to

bacterial cell wall. Receptors mediated attachment of opsonized bacteria has been the

recognization step of Phagocytosis.

2. Engulfment:

Leukocytes are able to respond to opsonins for that they display leukocyte

binding sites, once an opsonin coated particle is bound to the surface receptors of the

phagocyte, the particle is readily engulfed. Binding of a particle to the phagocytic

membrane elicits the engulfment, the phagocytic membrane flows around the particle

to enclose it in a cytoplasmic phagosome. Granular appearing lysosomes that contain

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DISEASE REVIEW

destructive agents then merge with the phagosome membrane, thereby bringing their

contents in to contact with the particle. As lysosome merge with the phagosomes their

number within the phagocyte are reduced and the phagocyte is degenerated. If the

particle is too large to be easily engulfed i.e. a multicellular parasite. There may be

regurgitation of the granule contents in to the tissue spaces. The leukocytes attempting

to engulf this large surface experiences frustrated phagocytosis and releases toxic and

degradative substances that damage the basement membrane and the surrounding cells

and the matrix.

The neutrophil cytoplasm contains two types of granules Azusophil and

Specific granules. Lysosomes contain acid hydrolases, neutral proteases, cationic

proteins, lysozyme.

i) AZUSOPHIL:

These lysosomes contain acid hydrolases, neutral proteases, cationic proteins,

lysozyme.

ii) SPECIFIC GRANULES:

Which contain lysozyme and lectoferiin but no hydrolases or peroxides.

Macrophages also contain azusophil granule. The process of degranulation pour in to

the phagosome powerful enzymes which kill the bacteria by different mechanisms.

3. KILLING AND DEGENARATION:

The micro-organisms after being killed by antibacterial substances are

degraded by hydrolytic enzymes, their mechanism fails to kill and degrade some

bacteria.These are of mainly two types, Oxygen dependent and Oxygen Independent.

1. Oxygen dependent:

It is an important mechanism of microbicidal killing by the production of

reactive oxygen metabolites (O2 H2O2, OH, HOCL, HOL, HOBr) a phase of increased

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DISEASE REVIEW

oxygen consumption by activated phagocytic leukocytes in presence of NADPH

oxidase.The NADPH oxidase which is present in the cell membrane of phagosome

reduces oxygen to superoxide ion (O2).

2O2 NADPH 2O2 (superoxide anion)

2O2 + 2H─ H2O2

Suproxide is subsequently converted in to Hydrogen peroxide (H2O2) has

bactericidal properties, this bactericidal activity is carried out either enzyme

myeloperoxidase present in the granules of neutrophils and monocytes or the enzyme

myeloperoxidse acts on Hydrogen peroxide in the presence of halides (Chlorides,

Iodide, Bromides) to form hypophalous acid (HOCl ). Which is more potent

antibacterial agent than hydrogen peroxide.

Reactive oxygen metabolites are useful in eliminating microbial organisms

that grow within phagocytes.

2. Oxygen Independent:

This mechanism involves the release of substances that damage bacterial cell

walls, disrupt bacterial replication and produce a low pH within the phagosomes

resulting from accelerated glycolysis, Which may be directly toxic and may indirectly

aid the function of other enzymes.

b) GENERAL RESPONSE:

The general celleular respons may be Fever and Leukocytosis.

i. Fever:

Fever is one of the most prominent systemic manifestation, particularly in

inflammation associated with spread of organisms into the blood stream. The patient

may have high fever charecterised by dramatic swings in the temparatur. The origin of

the fever may be uncertain, although it may be caused by the release of bacterial

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DISEASE REVIEW

endotoxins. In addition interleukin – 1 (IL-), prevously described as endogeneous

pyrogen released from the leukocytes is an important mediator of hyper pyrexia. This

mediator is taken up by lymphatics from the site of imflammation, which is circulated

in blood stream. It is believed that IL-1 initiates fever by inducing the synthesis of

PGE2 in the anterior hypothalamas and stimulating thermoregulatory centres.

ii. Leukocytosis:

Increase in the number of circulating white cell is another charecteristic

significance of acute and chronic inflammation. When the inflammation is deep and

the local symptomatology is either not observes or impossible to demonstrate, then

leukocytosis is of assistance in determining the severity of the infection and the

degree of the resistance offered by the body. This requires not only an absolute count

(i.e. the total number of white cells per mm) but also the relative number of each type

of leukocytes particularly the number of neutrophils. In making the differential count,

the number of each kind of white cells in hundred is counted.

• A high absolute count (T.L.C) with a high neutrophil percentage indicate

severe infection and good body resisitance.

• A high absolute count with a moderate neutrophil percentage indicates a

moderate infection and good body resisitance.

• A low absolute count with high neutrophil percentage indicates severe

infection and weak resisitance of the body.

• All inflammatory states do not evoke neutrophilic leukocytosis. Infectious

mono nucleoses, whooping cough, mumps, rubella and undulent fever

charecteristicaly produce lymphocytosis.

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DISEASE REVIEW

Allergic inflammatory reaction like hay fever, bronchial asthama and parasitic

infection typically elicit eosinophilia. Some infections may cause leukopenia instead,

like infections caused by viruses, rickettsiae, protozoa and salmonelloses.

C. TISSUE CHANGES OR REPAIR:

There are two types of tissue changes

a. Degenerative

b. Proliferative.

a. Degenerative:

The two most common degenerations are

1. Albuminous degeneration or cloudy swelling

2. Fatty degeneration

1. Albuminous degeneration or cloudy swelling:

It is closely related to hydrophic or vascular degeneration. Being manifestation

of a disturbance of protein metabolism, is called as Albuminous degeneration. It may

be caused by the bacterial toxins, chemical poison, malnutrition and other

disturbances.

The principle organs showing cloudy swelling are Kidneys, Liver and Heart

muscles. The organ affected is slightly enlarged owing to swelling of the cells of

which it is composed. It is pale as blood vessels being compressed by the swollen

cells. The cut surface has rather cloudy appearance and slightly opaque.

2. Fatty degeneration:

Fatty degeneration or Fatty metamorphosis is a true sickness of cells caused

by some injurious influences. It is best seen in Liver, Kidney and Myocardium. The

fat metabolism is interferred with fat accumulations in the cell. In some cases there is

merely unmarking of fat already present. The causes of fatty degenerations are

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DISEASE REVIEW

• Poisons

• Anoxia

The organs look fatty. The liver and kidneys are pallor and softer than normal.

The heart is soft and flabby. Ultimately the tissue becomes necrosed.

If the irritant is intense, the effect is degenerative destruction. If it is mild, acts

as stimulant and leads to proliferation. At the centre of the inflammatory area the

action of the irritant is severe, degeneration predominates at the periphery and the

action is mild. The tissue may be stimulated to proliferation. thus this part of the

inflammation is known as repair or healing.

i) SUPPURATION:

If the dead tissue in an inflammed area undergoes softening and liquification is

known as the process of Suppuration, the fluid formed is called the Pus, by this

method the dead material is removed from the body.There are three requisites for

suppuration.

i. Necrosis

ii. Presence of sufficient leukocytes

iii. Digestion of the dead materials by protolytic fermentation.

If any one of these are absent suppuration does not occurs.The digestive

enzymes are produced mainly by the leukocytes to a lesser extent by the necrosed

tissue cells and the infecting bacteria. These are neutralized by anti enzymes present

in the serum. If there is less leukocytes or more serum liquification does not take

place.

ii. ABSCESS:

It is an example of a localized suppuration. The inflammation is limited to the

area and as the irritant is pyogenic, pus is produced. The cells in the centre of the

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DISEASE REVIEW

inflammatory area are killed and liquified by protolytic enzymes. In this way a cavity

is produced which contains pus. The wall of the abscess cavity consists of damaged

but still living tissues. Hence the spread of infection is limited, this limiting zone is

crowded with polymorph nucleus leukocytes and macrophages filled with debris. Pus

cells are continuously dicharged from this, thus the abscess is chronic or if the

infection is dying out, the macrophages will greatly outnumber the polymorphs

further out, the tissue become more and more normal.

If the infection is continuously active more and more material is added to the

abscess, So that the pressure within the abscess increases. Thus it points in the

direction of less resistance. If the abscess enters the muscle sheath, it may extent

along a considerable distance.

iii BOIL:

It is an abscess of a Sebaceous gland or a hair follicle caused by the

Staphylococcus aureus, which has penetrated the opening of the duct. There is a

marked fibroblastic proliferation, which with intercellular formation of fibrin, causes

the characteristic indusation. The tension becomes high and causes pain. There may

be a very little liquification of the necrosed tissue, So that the centre of the boil is

composed of a solid "Core" instead of Pus.

iv. CARBUNCLE:

It is the infection spread to the subcutaneous tissue where it causes a more

diffused lesion which discharges on the surface by a series of openings. The pus

becomes inspected and the dead tissue is converted into a mass of fatty debris in

which lime salts may be deposited.

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DISEASE REVIEW

v. CELLULITIS:

When the suppuration spreads through the tissues, the condition is called

cellulitis. The fibrin that forms as a result of proliferation, inflammed part limit the

infection.

3. REGENERATION:

This implies to a complete renewal of the tissue as in liver. The process of

healing if fundamentally the same in all the wounds. It consists of two parts. Removal

of inflammatory material and necrotic debris, which may be more or little.

Replacement or reconstruction of the original tissue to a greater degree as much as

possible.

The repair involves the invasion and replacement of dying and dead tissue by

immature mesenchyma called Granulation tissue, which is a highly vascular tissue.

On account of its cellularity a granulating surface has a remarkable power of

resistance against bacterial infection. The granulation tissue grows to maturity from

below to up words. When the wound is aseptic the epithelium will grow in from the

edges as a delicate blue pellicles, gradually it becomes thick and opaque.

When the surface is covered by epithelium, the process of devascularization

begins. The new vessels, being no more needed will gradually disappear. The scar that

is red becomes white and bloodless.

8. REVIEW OF ANTI-INFLAMMATION

Drugs which can normally be used to almost every type of inflammatory

conditions are known as anti-inflammatory drugs These have two major groups.

1. Steroidal anti-inflammatory drugs in the form of gluco corticoids.

2. Non-Steroid anti-inflammatory drugs (NSAID)

Mode of action of steroidal anti-inflammatory drugs

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DISEASE REVIEW

ACTH and Glucocorticoids prevent the clinical features of inflammation i.e.

local heat, redness pain and swelling. Their action is based on reducing the increased

permeability of capillaries and maintenance of the integrity of the cell membrane

even in the presence of toxins. Stabilization of lysosomes release from the

granulocytes by inhibiting phagocytosis. Also acts on fats phase of inflammation and

inhibits capillary proliferation deposition of collagen cicatrisation. But fibrous tissue

once formed is not dissolve by corticosteroids.

Hence the anti-inflammatory effect of steroidal therapy is non specific. The

steroid inhibit the phospholipase enzyme which is essential to activate the cell

membrane. This activation release the arachidonic acid, which metabolises to produce

inflammation. But once the chain is initiated it cannot be stopped by the steroidal

therapy, moreover in large doses, it may interface with wound healing.

9. CLASSIFICATION OF ANTI-INFLAMMATION

These are broadly classified into two groups. Narcotic Analgesics or opoid

analgesics and Non-narcotic analgesics or non opoid analgesic or NSAID, non

steroidal anti-inflammatory drugs.

1. NARCOTIC ANALGESICS:

These agents are capable of relieving severe degree of pain but are moderately

or strongly addicting. This group includes opoids which binds to the opoid receptors.

These, further classified in to Natural opium alkaloids ( e.g. Morphin)., Semi-

synthetic opiums (e.g. Diacetyle morphin or acetyle morphin) and Synthetic opoid

(e.g. Pethedine).

2. NON-NARCOTIC ANALGESICS OR NON OPOID ANALGESIC or

NSAID:

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DISEASE REVIEW

In this the agents relieve mild to moderate degrees of pain and are considered

as non active. This group includes aspirin analgesic and other analgesics and

antipyretic drugs and these have no affinity for the opoid receptors, but the site of

action is only peripheral and further NSAID is divided in to Potent anti-inflammatory

and good analgesics, Potent anti-inflammatory and good analgesics, Moderate anti-

inflammatory and moderate analgesics and Poor anti-inflammatory and good

analgesics.

A. POTENT ANTI-INFLAMMATORY AND GOOD ANALGESICS:

i. Salcylates e.g. Aspirin

2. Oxy cum derivatives e.g. Paraoxicam.

B. POTENT ANTI-INFLAMMATORY AND GOOD ANALGESICS:

i. Pyrazolin derivatives e.g. Phenyl butazone.

ii. In dol derivatives e.g. Indomethacin.

3. MODERATE ANTI-INFLAMMATORY AND MODERATE

ANALGESICS:

i. Propyonic acid derivatives e.g. Ibuprofen

ii. Anthranlic derivatives e.g. Mefenamic acid.

iii. Aryl acetic derivatives e.g. Diclofenac.

4. POOR ANTI-INFLAMMATORY AND GOOD ANALGESICS:

i. Para amono phenyl derivatives e.g. Paracetamol or acetaminoidene.

ii. Pyrozolon derivatives e. g. Metamezole.

10. MANAGEMENT OF INFLAMMATION AND ACTION OF NON-

STEROIDAL ANTI INFLAMMATORY DRUGS (NSAID)

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DISEASE REVIEW

As stated earlier, the activation of cell membrane releases arachidonic acid, it

is metabolized by cyclo-oxygenase pathway and produce prostaglandins (PGs).These

include PGG2, PGH2, PGl2, PHE2 and PGE2 and cause vasodilatation and potentiate

oedema. Some of these are supported to inhibit platelet aggregation.

These further sensitize the chemical receptors of the afferent pain endings to

other chemical mediators like bradykinin and histamine. Aspirin and asperin like

NSAID have been shown to inhibit release or synthesis of PG5 and thus produce

beneficial anti-inflammatory condition where PGs are synthesized locally. It is unable

to produce any analgesic effect where the sensory nerves are directly stimulated.

Some of the NSAID produce anti-inflammatory effect indirectly,like

indomethacin. It inhibits phosphodiesterase and thus increases the intracellular

concentration of cyclic ATP, Cyclic ATP has been shown to stabilize membrane

including lysosomal membrane in polymorphonuclear leukocytes. This prevent the

release of enzymes important in the inflammatory responses. Some drugs may inhibit

the activation of T-lymphocytes which release lymphokinase, which plays an

important role in mediating inflammation.

9. REVIEW ON SHOTA

Nidana mean the causative factors which lead to the disease. nidana

parivarjana is the first line of treatment. According to different classics the nidanas of

shota can be classified as follows:

1. Ahara sambhandhi

2. Vihara sambhandhi

3. Anya.

1. Ahara sambhandhi: Abhakta, Gunaheena Bhojana sevana by krisha and

durbala person, kshara, Amla teekshana ushna, Guru padartha Adhika

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DISEASE REVIEW

sevana,Dadhi, Apakwa Bhojana, Mrittikia, Shaka, Viruddha Bhojana, Dushta

Bhojana, Gara Visha.

2. Vihara sambhandhi: Nishkriyata, Ashodhita,

3. Anya: Marmopaghata, Vishama Prasooti,

POORVAROOPA

Daha, Engorged Veins, Anga gourava.

ROOPA

Roopas are those which are well manifested by which diseases are diagnosed.

The samanya laxana of shota are as fallows-

Sagourava

• Anavashtitatwa

• Ushnata

Utsedha Wherever there is utseda it is considered as shota.

NIDANA

• Siratanutwa

• Saloma harsha

• Vivarnata

BHEDA

All the shothas are of single type but according to hetu vishesha they are again

divided in to different types.

Charaka in his chikista sthana has mentioned seven types of shotha. They are

as fallows:

1. Vataja

2. Pittaja

3. Kaphaja

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DISEASE REVIEW

4. Dwandaja

5. Sannipataja

6. Abhigataja

7. Vishaja.

Where as Susruta has mentioned only five types of shotha, they are as follows:

1. Vataja

2. Pittaja

3. Kaphaja

4. Sannipataja

5. Vishaja.

Vagbhata in his Nidana sthana has mentioned Seven types of shotha

1. Vataja

2. Pittaja

3. Kaphaja

4. Dwandaja

5. Sannipataja

6. Abhigataja

7. Vishaja.

Where as in Madhava nidana we get again seven types of shotha

1. Vataja

2. Pittaja

3. Kaphaja

4. Dwandaja

5. Sannipataja

6. Abhigataja

7. Vishaja.

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DISEASE REVIEW

Table showing the different types of shota according to different classics.

Table no:8

Bhedha C h S u V a M n

Vataja + + + +

Pittaja + + + +

Kaphaja + + + +

Dwandaja + _ + +

Sannipataja + + + +

Abhigataja + _ + +

Vishaja. + + + +

Looking the types of Shothas according to the different classical texts, we

come across similarity in view of Charak, Vagbhata and Madhava nidana i.e. the

Seven types of shothas are explained, where as in Sushruta we get only five types of

shotha.

SAMPRAPTI

Vata gets vitiated with its own vitiating factors which carries the rakta, pitta

and kapha to utthana siras which gets obstructed there and settles in between the twak

and mamsa. This sanchaya of rakta, pitta causes utseda which results in shotha.

SADHYASADHYATA

Before going to start the chikista of a particular disease, one should know

about the sadhyasadhyata of a disease, i.e. whether it is easily curable, with efforts or

incurable etc should be known. According to prabhava, the diseases are classified as

sadhya and asadhya. Sadhya is subdivided a sukha sadhya and krichra sadhya where

as asadhya is subdivided as yapya and pratyakhya.

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DISEASE REVIEW

If the rogi is krisha or Durbala or if associated with upadravas, when it moves

to the marma sthana, ruja yukta and associated with srava and sarvangashotha is

considered to be asadhya. Where as in aheenamamsa, ekadoshaja, nava, balavan

vyakti shotha is sukha saadhya.

UPADRAVA

Chardi, trishna aruchi,swasa, jwara, atisara,dourbalya, pipasa, hikka, kasa are

the updravas of shotha.

CHIKITSA SOOTRA

Depending on bala, dosha and kaala, one has to see the nidana, dosha, rutu and

viparita chikitsa has to be done.

CHIKITSA

Samanya chikitsa

1. In ama dosha yukta firstly langhana and pachana

2. In bahu dosha avastha shodana

3. Shotha in adha pradesha-virechana

4. Shotha in sira pradesha shiro-virechana.

5. Shotha in urdhwa pradesha vamana

6. If shotha has occurred due to excess sneha vastu sevan then ruksha kriya is to

be done.

7. In vataja shotha if vibhanda of mala is present niruhabasti to be given.

Oushadhis used in Shotha

1. Gandiradyarishta

2. Astashataarista

3. Punarnavadyarishta

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DISEASE REVIEW

4. Phalatrikaadyarishta

5. Kshragutika

6. Kamsaharitaki

7. Chitrakaghrita.

PATHYA

Kulatha yusha, Trikatu, Yavakshara choorna, Mudga, Vishakeira, Jangala

pashu pakshi mamsa, Koorma, Shiki, Shallka mamsa rasa, Sauvarchala, Grinjana,

Patola, Vayasi, Moolaka, Vetra, Nimba, one year old Yava and Shaali dhanya.

APATHYA

Gramya, Jaleeya, Anupa mamsa, Lavana, Shushka, Navanna, Gouda (Guda

Padartha), Pishtanna, Dadhi, Tila, Vijjala, Madya, Amladravya vallura, Samashana,

Guru,Asatmya, Vidahi anna, Divaswapna and Maithuna.

54

MATERIALS AND METHODS

MATERIALS AND METHODS

A) Pharmacognostical study:

Aim: The aim of present study was to see Morphological, Microscopical and

determination of pH of Panchanga of Kakamachi.(Solanum nigrum linn.).

1) MORPHOLOGICAL STUDY:

Materials: The materials collected for the studies were.

Drug: Solanum nigrum linn. (Kakamachi)

Parts: Leaves

Collection of Materials: The leaves of solanum nigrum linn were collected freshly

from local viscinity.

Equipments: Sense organs.

Chemicals: Formalin Aceto alcohol (F.A.A), Chloralhydrate, Conc HCL.

Methods:

i) Organoleptic Method ii) Extra features.

i) Organoleptic Characteristics: In this method nature of the leaves, colour, taste,

size, shape, odour, were studied with the help of sense organs.

ii) Extra Features: The arrangement of leaves and its special characters were

studied.

2) MICROSCOPICAL STUDY:

Materials: The materials collected for the study were.

Drug: Fresh leaves and fine powder of leaves of Solanum nigrum linn.

Equipments: Compound microscope, eye piece, camera lucida, Glass slides, cover

slips, watch glass, camel brush, mountain brush, filter paper, blades, spirit lamp,

pipettes.

Chemicals: Fluroglycenol, Chloral hydrate, Conc HCL, Glycerin and Iodine.

55

MATERIALS AND METHODS

Methods: 1) Section Method 2) Staining process method.

1) SECTION METHOD:

• Put the sample in a test tube add sufficient chloral hydrate solution.

• Hold the sample vertically in between the thumb and fore finger

• With the help of a new blade, a thin transverse section of the sample is taken

• 10-15, thin, sufficient sections were taken .Thick and oblique sections are

rejected.

• With the help of mountain brush the sections are transferred to a watch glass

containing water.

2) STAINING PROCESS:

• Transverse section of the sample was taken and transferred it on to a slide with

the help of mountain brush

• Add a few drops of water

• Add a few drops of chloralhydrate solution and allowed to heat for two to

three minutes.

• Add equal proportions of fluro-gylcenol and Concentrated Hydrochlroic Acid,

warm gently on a flame and cool it , finally add a drop of glycerin and cover

the section carefully with coverslip, focus the section under microscope and

the arrangements of cells were studied.

MATERIAL:

Drug: Solanum nigrum linn.

Parts: Leaves (Coarse powder) and thin sections of leaves.

Equipments: Compound microscope, eye piece, camera lucida, Glass slides, cover

slips, watch glass, camel brush, mountain brush, filter paper, blades, spirit lamp,

micro meter.

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MATERIALS AND METHODS

CHEMICALS:

Fluro-glycenol, Chloral hydrate, Conc HCL, Glycerin.

METHODS:

Stomatal number, Stomatal index, Veinislet number, Vein termination

number, Palisade ratio.

i) Vein Islet and Vein Termination:

METHODOLOGY:

• 3-4 pieces of fresh or dried leaf are cut from the middle portion of the lamina

avoiding midrib and margin.

• These are taken in a test tube and boiled with chloral hydrate solution in a

water bath, until they are clean enough for observation.

• Different cleaning methods are applied for individual leaves that mainly have

very thick lamina

• The pieces are taken on a watch glass and mounted on a glass slide in chloral

hydrate solution with lower surface of the leaf facing upwards so that the veins

which are more prominent in the lower surface are seen clearly under the

microscope.

• For this study 6x eye piece and low power objectives were used. The stage

micrometer is focused (1 mm) and the camera lucida is fixed in such a way

that the aperture of it is in the same line with that of the eye piece.

• Black drawing sheet was placed on the same side of the microscope where

camera lucida is fixed.

• Using a white pencil the first and the last of the stage micrometer was

removed and the slide was mounted with the leaf specimen and focused in the

same way.

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MATERIALS AND METHODS

• The square drawn in the paper was adjusted in such a way that it lies exactly in

the middle of the field of vision and the image of the leaf piece mounted

appears to be superimposed on the square of the drawing sheet.

• Starting from one side all the vein islets inside the square as well as on the

boundary were traced. The vein termination within the square only was taken

in to account.

ii) STOMATAL NUMBERS:

The stomatal number is a very specific criteria for identification and

characterization of leafy crude drugs.

METHODOLOGY:

• Stomatal number is the average number of stomata per mm2 of epidermis and

the number on each surface of a leaf. Each stomata consist of two guard cells

and the spore is counted as single unit.

• It is indicated by the symbol ' S '

iii) STOMATAL INDEX:

• Stomatal index is defined as the percentage of stomata from the total number

of epidermis cells, which can be explained as

S= The number of stomata in a given per unit area of leaf.

E = Total number of epidermal cells including trichomes in the area of leaf.

METHODOLOGY:

• Fragments of leaf of 5x5 mm2 in size were taken in a test tube containing

about 5 ml of chloral hydrate and heated on a water bath until the fragments

are transparent, using the same procedure as described earlier in veinlet

termination number determination.

58

MATERIALS AND METHODS

• A peeling was easily taken by partially cutting one of the veins with a blade on

the lower epidermal region and pulling it.

• This membranous layer which came out along with the vein was taken on a

glass slide by cutting the thick vein section attached to it.

• A few drops of chloral hydrate solution are added and the cover slip is placed

on top. The peeling was sufficiently big enough to cover the entire field of

view when mounted, A 10x eyepiece and high-powered objective were used. 1

mm2 of area is taken with the help of a stage micrometer as described in the

previous section. On the tracing paper a circle smaller than that of the field of

circle/square is clearly visible in the centre of the field of vision.

Stomatal Index = S x 100 E + S

iv) PALISADE RATIO:

METHODOLOGY:

• Small pieces of leaf from the apex taking the middle and basal portion of the

lamina, were taken from the leaves, the lower epidermis was peeled off and

then cut into pieces.

• It was boiled gently in a test tube taking 4-5 leaf pieces in about 5 ml of

strong solution of chloral hydrate till the green colour disappears. Then the

pieces were kept separately on a glass slide with its upper epidermal layer kept

upper most.

• It was focused under high power objective with 15x eyepiece. Four clear

continuous epidermal cells were observed and then the fine adjustment knob

of the microscope is turned down slowly to observe the palisade cells.

• After tracing four continuous epidermal cells, moving the fine adjustment of

the microscope, palisade cells inside all the 4-epidermal cells were drawn.

59

MATERIALS AND METHODS

• The area of the sample was changed and the experiment should be repeated to

those that are 50 % or more inside the outer boundary of 4 epidermal cells are

taken in to account.

• The number of total palisade cells were divided by 4 which give the average

number of palisade cell under each epidermal cell.

v) pH Value:

EQUIPMENTS:

A pH meter is used to analyze and measure the pH having a reading scale

extending from 1 to 14.

Methodology: By electro metric method

• In this method hydrogen electrode may be used to measure hydrogen ion

concentration and also pH

• The potential, which is created between electrode and the solution is

reproducible and its magnitude depends upon the concentration of hydrogen

ion.

• A constant electrical potential against which the potential of the glass

electrode can be measured is provided by the reference electrode. This consists

of a metallic internal element immersed in a electrolyte saturated solution of

KCL.

PHYTOCHEMICAL STUDY:

Aim:

The main aim of the Phytochemical study was to know the chemical

constituent in a trial drug, subjecting to different tests like extraction, preliminary

phytochemical tests and isolation of extracted fractions characterized by T. L. C, U .V

and I. R method.

60

MATERIALS AND METHODS

SOLUBILITY OF SOLANUM NIGRUM LINN. :

Materials: Fine powder of panchanga of Solanum nigrum linn. (Kakamachi)

Solvents:1) Petroleum ether, 2) Ethyl Acetate ,3) Chloroform, iv) Ethyl alcohol etc

METHODOLOGY:

Taken in to the different filter paper in different funnels according to the

different solvents after seeing residue which was minimum that solubility of the drug

in that solvent is maximum.

1) EXTRACTION:

MATERIALS:

Drug: Coarse powder of panchanga of Solanum nigrum linn. (Kakamachi)

Equipments: Soxhlet apparatus of 1,000 ml, round bottom flask, water condenser with

distillation apparatus, beakers 500 ml measuring cylinder, thermostat, electronic

weighing machine, filter paper, magnetic stirrer, boiling chips etc.

Chemicals: 90 % ethyl alcohol

Methods: The air dried panchanga of Solanum nigrum linn. drug was subjected to

exhaustive extraction, by soxhlet apparatus by around 18 hours with 90 % ethyl

alcohol, extraction was done in three batches of these , one batch of coarse powder

with ethyl alcohol. The extraction process is carried out for about 18 hours to each

batch. After the extraction the solvents were distilled off to obtain semi solid extract

and concentrated on magnetic stirrer, the weights of each batch of extract were

recorded.

61

MATERIALS AND METHODS

2) Preliminary phytochemical tests:

MATERIALS;

Drug: Extractive sample of Solanum nigrum linn.(Kakamachi)

Equipments: Test tube with holder, stand, spirit lamp, pipette, glass rods, beaker 50

ml to 250 ml, conical flask, waterbath, burner with stand.

Chemicals: 10% conc H2SO4, chloroform solution, acetic unhydride, sulphur powder.

soda lime, million's reagent, mercuric sulphate 10%, Sulphuric acid 1%, sodium

nitrate 5%, sodium hydroxide 1%, copper sulphate , 10% tannic acid, acetyl chloride,

zinc chloride, Mayer's reagent(Saturated picric acid) Solution, Dregendroff's reagent(

Potassium bismoth iodide) Ammonium renicate, Molish's reagent, Barford's ,

benedit's reagent, saponin, ferric chloride, fragments pieces of magnesium ribbon and

concentrated hydrochloric acid, zinc dust, sodium hydroxide, 10% lead acetate,

Bromine water etc.

Methods:

(i) Test for sterols:

(a) Salkwoski’s test: A few drop of concentrated 10% sulphuric acid was added

to the 5 ml sample solution, shaken and allowed to stand.

(b) Liebermann- Burchardt test: The sample solution 5 ml of extract mixed

with few drops of acetic unhydride. Where concentrated 10% sulphuric acid

(H2SO4) sulphuric acid (H2SO4) was added from the sides of the test tube.

(c) Sulphur test: Sulphur when added to the extract, it sinks in it showing the

presence of sterols.

Preparation of test solution:

Dissolve 0.5 ml of test solution in 100 ml of water by heating and using the

solution for following test.

62

MATERIALS AND METHODS

(ii) Test for Proteins:

(a) Test solution + Soda lime and heat

(b) Test solution + Millon’s reagent mixed and allowed to stand

(c) 1 ml test solution + 1 ml 10% mercuric sulphate in 10% sulphuric acid.

Boiled gently for 30 seconds added 2 drops of 1% sodium nitrite solution.

(d) Biuret test: 3 ml test solution + 1 ml 5% sodium hydroxide with + 2 drops

of 1% CuSO4 solution mixed and allowed to stand.

(e) Test solution + few drops of 10% tannic acid mixed and allowed to stand.

(iii) Test of Triterpenoids:

(a) Salkwoski’s test: A few drops of 10% concentrated sulphuric acid is added

to the 5 ml of sample solution, shaken and allowed to stand.

(b) Liebermann’s – Burchardt test: The sample solution mixed with few drops

of acetic unhydride, in which concentrated sulphuric acid (H2SO4) is added

from the sides of the test tube.

(c) “Tschugajew test ”: Where excess of acetyl chloride and a pinch of zinc

chloride are added to the sample containing, kept aside and warmed on

water bath.

(iv) Test for alkaloids:

(a) Mayer’s test: The sample solution with mayer’s reagents ( potassium

mercuric iodide) mixed and allowed to stand

(b) Wagner’s test: sample solution with wagner’s reagents (iodine in

potassium iodide) mixed and allowed to stand.

(c) Hager’s test: sample solution with Hager’s reagents (Saturated picric acid)

mixed and allowed to stand.

63

MATERIALS AND METHODS

(d) Dragendroff’s test: Sample solution with Dragendroff’s reagent (

Potassium Bismuth iodide) mixed and allowed to stand.

v) Test for Carbohydrates:

a) Molisch test: sample solution with few drops of molisch reagent and 2 ml of

concentrated 10 % H2SO4 added slowly to the sides of the test tube.

b) Barford’s test: sample solution with Barford’s reagent boiled on a water

bath.

c) Benedict’s test: sample solution with Benedict’s reagents and boiled on a

water bath.

vi) Test for Saponin’s:

(a) Foam test: sample solution mixed with saponins and shaken, shows

formation of froth.

(b) Haemolysis test: 2 ml of 18% sodium chloride solution in two test tubes

was taken to one test tube added distilled water and to the other test tube 2

ml of sample solution was added few drops of blood is added to both the

test tubes.

(vii) Test for flavonoids:

(a) Ferric chloride test: sample solution with few drops of Ferric chloride

solution mixed and allowed to stand.

(b) Schinoda test: Sample solution with few fragments of magnesium ribbon

and 10% concentrated hydrochloric acid mixed and allowed to stand.

(c) Zinc-HCl reduction test: Sample solution with zinc dust and few drops of

HCl mixed and allowed to stand.

(d) Alkaline reagent test: Sample solution mixed with sodium hydroxide.

64

MATERIALS AND METHODS

(e) Lead acetate test: Sample solution mixed with few drops of 10% lead

acetate.

(f) Bromine water test: Sample solution mixed with bromine water and

allowed to stand.

(viii) Test for tannins:

(a) 1 ml of sample solution when mixed with ferric chloride and allowed to

stand.

(b) Sample solution mixed with 10% lead acetate and allowed to stand.

(c) Sample solution mixed with bromine water shows yellow colour.

Identification by T.L.C.:

Drug: Extraction of sample, which is treated with 1:10 ml solute: solvent like ethyl

alcohol with dilution method.

Equipment: Silica gel, T.L.C. kit, hot air oven, standard glass, Wattaman glass plate,

beakers, sprayer.

Chemicals: Dragendroff’s reagent, silica gel, ethyl alcohol.

METHODS:

T.L.C. of the ethyl alcohol extract of the sample was carried out as follows:

The silica gel powder mixed with water and made thin paste, then with the

help of glass slide, the silica gel was spread on glass plates uniformly. After

sometimes the air dried plate were kept in a hot oven at 1100C – 1200C heat was given

continuously, then the prepared sample is kept on a side of the plate then immersed in

solvents upto 30 minutes then Dragendroff’s solution is sprayed on the plates.

A parameter called the Rf value is always used in TLC this is determined as

follows;

Rf = Distance from the base line to the spot Distance from the base line to solvent

65

MATERIALS AND METHODS

Materials for partial characterization by U.V. and I.R. spectroscopy:

Drug: Extractive alkaloid sample of Panchanga of Solanum nigrum Linn.

Equipments: Spectroscope potassium bromide disc.

Chemical: Ethyl alcohol.

METHODS:

• Different chemicals when subjected for photometry in white light (including

U.V) have specific affinity to absorber to transmit particular range of

wavelength, which is related to that compound.

• Spectrophotometer analysis involves the measurement of the ability of the

dissolved solutes to absorb light of definite and narrows wavelength ranges.

• These absorption are measured at a wavelength that are generally a

characteristic of the chemical composition of a dissolved absorbing substance

radiant allergy waves range from 200 nm to about 380 nm in the UV region

and from 380 to around 780 nm in the visible region.

• The UV or visible spectrum of a molecule is the result of change in energy of

a molecule as shown or rather than of a particular bend. The UV and visible

spectrum of a substance generally not have a high degree of specific but they

are suitable for quantitative essays for many substances and useful as

additional means of identification.

• Hence, the UV spectral analysis was selected as one of the parameter. The UV

visible spectra of the sample was recorded in a schematized double beam UV

visible recording spectro-photometer (Model UV – 160A).

UV Spectrophotometer analysis:

The alkaloid fraction was subjected to spectral analysis. The details of spectral

analysis are as follows;

66

MATERIALS AND METHODS

The UV absorption spectrum of the alkaloid was recorded on schematize

model 120 at medium. Scan speed the spectrum has shown a strong peak at 140 nm

and a shoulder peak at 260 nm.

UV spectrum:

Ethyl alcohol

(i) 240 nm (strong)

(ii) 260 nm (shoulder) Max

IR Spectrum:

The IR spectrum of the alkaloid was recorded on perkins Elumesk model 183

at medium scan speed by applying potassium bromide disc. Peak at 3679.90 indicates

N = H stretch.

And peak at 1518.68 indicate C = 0 stretch

Peak at 627.27 indicate C – bond Max KBr disc

(C) Experimental study:

Aim: To evaluate the effect of extracted fraction of Panchanga of Solanum nigrum

linn by inducing to the albino rats to see its anti-inflammatory activity.

(I) Selection of animals: Charles foster strain albino rats of either sex weighing

between 110 – 180 gms for experiments with the following conditions.

(1) The animals were obtained from the animal house attached to the medical

college laboratory.

(2) They were exposed to natural day and night cycles with ideal laboratory

condition in terms of suitable temperature and humidity. They were feeded

67

MATERIALS AND METHODS

with Amrut brand rat pallet feed which are soaked in oil and which are

supplied by Nava maharastra chakana oil mills and tap water.

(3) They were kept in a good cage and supplied the tap water.

(4) The rats being kept on standard diet to reduce the mortality of the rats and they

can be maintained.

(II) Sample size:

24 albino rats were taken for the experimental study distributed six in each

group.

(III) Preparation of the test drug and administration: Table : 9

Group I Standard

Group

Ibuprofen suspension was purchased and fed orally to the

albino rats at the dosage of 162 mg/kg.

Group II Trial

Group

The Solanum nigrum linn extracted was weighed and given

to trial group in minimum dose to the albino rats at the

dosage of 270 mg/kg.

Group III Trial

Group

The Solanum nigrum linn extracted was weighed and given

to trial group in maximum dose to the albino rats at the

dosage of 270 mg/kg.

Group IV Control

group

1% normal saline was fed orally to the albino rats at the

dosage of 1 ml each animal in the single dose.

(IV) Grouping: The rats were grouped into four groups of six in each group.

Group I: Standard group: Ibuprofen suspension in the dose of 162 mg/kg.

Group II: Trial group (minimum dose): Extraction of Panchanga of Kakamachi with

aqueous solution in the dose of 270 mg/kg.

68

MATERIALS AND METHODS

Group III: Trial group (maximum dose): Extraction of Panchanga of Kakamachi with

aqueous solution in the dose of 540 mg/kg.

Group IV: Vehicle drug group or observation group.

Preparation of Carragrinine:

Here no further preparatory methods followed. The Carragrinine taken in the

dosage 0.1 ml.

(V) Induction method of Carragrinine: Carragriine is taken for the observational

study purpose and this was given in the techniques hind rat paw oedema. At

the doses of 0.1 ml to each animal at single dose for experimental study, to get

the inflammation.

(VI) Dose selection:

The extracted fractions of Panchanga of Solanum nigrum linn was given with

aqueous solution in the doses of 270 mg/kg Carrageen was induced in a dosage of 0.1

ml in 150 mg/kg body weight to the albino rats.

• Trial drug to the albino rats (Minimum dose) = 270 mg/kg.

• Trial drug to the albino rats (Maximum dose) = 540 mg/kg.

• Standard drug to the albino rats = 162 mg/kg

• 1% normal saline water = 0.2 ml/200gms of rat.

Conversion to 1kg body weight:

Trial drug ( Minimum Dose) :

= Human dose X 0.018 i.e., 3 gm x 0.018

= 0.054 gm or 54 mg.

= 54 x 5 = 270 mg/kg.

Rat: 270 mg/kg.

69

MATERIALS AND METHODS

Trial drug ( Maximum Dose ):

= Human dose x 0.018 i.e., 6 gm x 0.018

= 0.108 gm. or 108 mg.

= 108 x 5 = 540 mg/kg.

Rat:540mg/kg.

Standard Drug:

= Human dose x 0.018 i.e., 1800 mg x 0.018

= 32.4 mg

= 32.4 x 5 = 162 mg/kg

Rat: 162 mg/kg

(VII) Route of drug administration:

The test drug was administered to the albino rats according to their body

weights of the animals by oral route with the help of infant feeding tube.

(VIII) Duration of treatment:

The test drug was given in minimum and maximum doses for Group II and III

respectively, standard drug Ibuprofen was given to Group – I and for Group IV 1%

normal saline was given to Carragrinine induced rats, later Anti-inflammatory effect

was evaluated.

Statistical analysis:

The data collected were statistically analyzed by using unpaired student ‘t’ test

with the consultation of bio-statistician.

70

OBSERVATIONS AND RESULTS

OBSERVATIONS

1) Observation of Pharmacognostical

The pharmacognostical study includes,

• Morphological study

• Microscopical study

• Physical evaluation of the drug

A) Morphological observation:- In this study the Morphological characteristics

were observed by Organoleptic method.

Table No : 10

Colour Green

Taste Tikta

Size 2.5-9 by 2.5 cms

Shape Ovate

Odour Characteristics acceptable

Nature of leaf Acuminate, glabrous, thin, entire sinuate toothed, tapering into the

petiole,

Touch Smooth

Beside these extra features, the arrangement of the leaves and their special

characteristics were observed such as

1. Lanceolate

2. Membranous

3. Long petiole.

71

OBSERVATIONS AND RESULTS

Macro morphological evaluation of KAKAMACHI:

While analyzing a herbal drug, an idea about the distribution of various cell

types within different plant organs is essential. After evaluation of the different

fragmented structures of a comminuted material, the charecterstics related to the

initial structures and thereby the identity of the original material can be revealed.

• In the intact material the examination of the tissue distribution is based on the

evaluation of different basic cell types and cell inclusion is described earlier is

important for identifying KAKAMACHI.

• Cytomorphological evaluation of KAKAMACHI is based on the examination

of the basic cell types and inclusions that may not be botonically complete, in

leaf where epidermis with stomata, cellulose, parenchyma, vascular eliments

and chlorophyll were frequently present structures include epidermal

trachomous glands and palisade cells, crystals of calcium oxalates, pericyclic

fibers and collenchyma. For differentiating closely allied leaves, the

determinations of differential characters like Vein Islet number, Stomatal

Number, Palisade Ratio and Stomatal Index play a major role.

72

OBSERVATIONS AND RESULTS

Shape of Leaf: Table No : 11

Shape Ovate

Length and Width 2.5-9 by 2.5 cm

Mid rib Present at the centre of the leaf

Glabrous Surface is free from hairs

Lamina Structure The lamina is the flat part of the leaves which constitute the major

portion in leaf drugs. It can show a very wide variation in its form.

Shape of Lamina In case of a dried leaf where the original shape of the leaf was

obscured it has to be soaked in warm water and spread. Here the

shape of lamina was ovate.

Composition of

Lamina

In leaves containing herbal drugs, they include true leaves of

individual leaflets of compound leaves. This can be easily

distinguished if the attachment of the leaf to the stem can be

examined. Various compositions of the leaf have been

imperipinnate, which depends on the presence of a terminal

leaflet.

Apex Acuminate

Base The lower extremity of the lamina, is symmetrical.

Venation Arrangement of the Veins on the lamina

Dorsiventral leaf These have a palisade layer below the upper epidermis and a

spongy mesophyll above the lower epidermis

Margin The margin of the leaf is entire, tapering sinuate toothed.

73

OBSERVATIONS AND RESULTS

B) Microscopical Observation:

• Microscopical study includes the examination of the cell form and

arrangement of the different cells in a drug. The KAKAMACHI were

generally used in powdered or comminuted form where the macro-

morphology is destroyed, so that the evaluation of the microscopical cell

characters was essential indication.

• The KAKAMACHI contain some basic cell types e.g. Parenchyma,

Collenchyma, Sclerenchyma, Xylem, Phloem, Epidermis, Periderm etc.. along

with some cell inclusion characteristics i.e. the presence of ergastic substances

like starch, calcium oxalate, Calcium carbonate, alueronic, silica and different

other cell contents. Analysis of the plant drugs based on the distribution of

these various cell types within different organs is important to ensure the

identification.

• The basis of analysis by evaluating microscopical characters was that there

were always sufficient differences in the same type or different type of plants

as far as the cell characteristics were concerned.

Standardization profiles of KAKAMACHI were not available for most drugs.

As with any comparative procedure, the more information that usually unique, were

of greatest value, as they constitute the difference between adulterants and evaluation

of the microscopical characters only can provide such information of a particular

drug can be stored and reviewed in various ways by section method and by staining

process method under compound microscope observed the following events such as

Parenchymal cells, Stomatal cells, Xylem, Phloem, Vascular bundles, Palisade cells,

Starch grains, Fibres, Tracheids, Vessels, etc were seen.

74

OBSERVATIONS AND RESULTS

A) Observation of Parenchyma:

• It occurs as general ground tissue in most plants. These are usually asymetric,

thin walled and simplest type of cells. By course of maturity they may have

intercellular spaces.

• Secondary thickening may be present in reticulate or pitted form. Which can

be lignified. The cytomorphology of different types of parenchyma has been

shown.

B) Observation of sclerenchyma:

This is the hard supporting tissue with heavy secondary thickening. They are

generally divided in to two categories in respect of their aspect ratio as follows

• They are roundly isodiametric, although elongated and branched form may

also occur.

• They are found singly or as a complete layer or in groups with pitting and

stratification often accompanying them.

• They occur in the hard outer coat of seeds and fruits and pericycle regions of

woody stem.

C) Observation of Fibres:

• They are thick walled with high length and width. Fibres are classified based

on the area in which they occur In pericycle, Xylem or Phloem fibres.

• Crystal sheaths are sometime formed , this feature together with different size

frequency and distribution plays an important role as a diagnostic feature. The

ground mass of secondary xylem of picroena excels in building up of

compactly arranged thick walled wood fibres.

75

OBSERVATIONS AND RESULTS

• The Secondary Xylem contains wood fibres arranged in bundles. The Phloem

fibres of liquorices resemble those of Xylem in being enclosed in a crystal

sheath

• The contribution, abundance, Size and shape of the phloem fibres constitute an

important characters for the differentiation of medicinal bark.

D) Observation of Collenchyma:

• It is a supporting tissue directly derived from Parenchyma with greater

mechanical strength. Secondary thickening is much more and composed of

cellulose. The thickening may be stratified or unevenly distributed around the

circumference of the cell. Collenchyma is present above and below the midrib

bundle in many leaves.

E) Observation of Xylem:

• This is the principle water conducting tissue of a plant. They have lignified

secondary thickened wall that can show a variety of forms. Secondary growth

in thickness of stem and root is usually accompanied by the formation of

secondary xylem.

F) Observation of Tracheids:

These are the basic cell type of xylem tissue with a lignified, thickened and

pitted cell wall, it takes the form of a water conducting cell in a plant.

G) Observation of Vessels:

These constitute the fundamental conducting elements of the Xylem in

angiosperm. The most primitive type of vessels consists of a vertical series of trachied

like segments where as the advance type of vessels show complete dissolution of the

end walls to give slit like opening. The essential difference between trachieds and

76

OBSERVATIONS AND RESULTS

vessels is that the former are imperforate, where as the latter have pores at the end

which are connected to form a continuous file or tube.

H) Observation of Xylem Parenchyma:

These cells are axially elongated, sometimes thin walled but often with

thickening and lignifications. It functions as a storage tissue and in some cases the

cells are blocked with starch.

I) Observation of Phloem:

This compound tissue is responsible for the transport of food. It contains

companion cells, Sieve tubes, Phloem parenchyma and secretary cells. The sieve tube

is the conducting element in phloem. The sieve elements are the highly specialized

cells in phloem and the main morphological characteristic is the occurrence of sieve

in their walls which may often be detected by recognition of callus pad which show

some staining characters (Chlor-zinc-Iodine solution stain callose) to a redish brown

colouration. Anilineblue stains in to callose blue solution of ammoniacal copper

nitrate (BP) does not dissolve cellulose.

J ) Observation of Epidermis:

• This consists of a single layer of cells covering the whole plant i e. the outer

most layer of the plant structure. The epidermis of the root constitutes the

pilferers layer, shoots contain a compact layer of cells and that in contrast to

the stomatal guard cells, are often devoid of chloroplast

• The epidermal cell of the two surfaces of a leaf differs in form. Various

diagnostic features including the shape of the anticlinal (Vertical) and

Periclinal ( Horizantal) wall (Straight or wavy), the presence of thickening and

occurrence of striations on the surface. Cuticle can play a major role in

detecting the epidermis.

77

OBSERVATIONS AND RESULTS

• Epidermis has a specialized structure and most universal of these are stomata

which control water loss from the plant. They occur most frequently on young

leaves and stem. The structures of the epidermis and stomata are of first

importance in the microscopical identification of leaves.

• Different types of stomatas on the basis of their arrangement have been

shown. Trichomes are the out growths of epidermal cells, which occur in all

parts of a plant.

• They are of the value in the analysis of KAKAMACHI with different

distribution and frequencies. They are particularly useful in the examination of

fresh material where the stomata and epidermal cells are not readily visible.

K) Observation of Periderm:

This is a protective tissue that replaces epidermis in stem and root. Typical

periderm is usually present in root, of aquatic and subterranean stems and in the aerial

stems of plants cork cambium on the inside has been shown.

L) Observation of Starch:

• This is the most common carbohydrate reserve and is found in varying amount

in almost all plants.

• It occurs in granules of varying sizes and found most abundantly in roots,

rhizomes, fruits and seeds where they occur as larger grains.

• The small granules are formed in chloroplasts by the condensation of sugar,

which afterwards hydrolyzed; so that they pass in to the solutions of storage

organs, where under the influence of leucoplasts. Large grains of reserve starch

are formed.

• Starch is considerable pharmaceutical importance. Various such starches include

maize.

78

OBSERVATIONS AND RESULTS

• Starches of different categories, between crossed polaizer the granules appear

bright on a black background and each usually shows a dark maltase cross due

to the structure of the granule.

• This type of appearance is completely specific to starch although it is shown by

the granules of insulin. Starch occurs as irregular, angular masses or as a white

powder. It is insoluble in cold water but forms a collidal solution on boiling with

15 parts(w/w) of water with it, on cooling this solution forms a translucent jelly,

while heating with water the granules first swell and undergo gelatinization.

• Starch granules also undergo gelatinization when treated with caustic potash.

concentrated solution of calcium or zinc chloride or that of chloral hydrate.

• For microscopical examination of starch in herbal starch solution in it. Starch is

identified by its characteristic appearance in natural and polarized light and by

the formation of bluish black coloured compounds (Starch Iodide) with N/50

iodine solution, where as insulin does not stain with this iodine.

• Starch granules usually contains two carbohydrates.

C) Observation of Physical evaluation:

After collecting the materials and studied, the physical properties were

observed as

1) Observation of Vein Islet:

• Vein Islet was the term used to indicate the minute of the photosynthetic

tissues encircled by the ultimate divisions of the vascular stands.

• A Vein Islet was the smallest unit of the tissue encircled by the ultimate

division of the conducting strands of the leaves. The area of leaf considered

was preferably taken from the lamina midway between the midrib and the

margin.

79

OBSERVATIONS AND RESULTS

• The number Vein Islet per square mm was termed as Vein Islet number. This

number per unit area of leaf was constant.

2) Observation of Vein let termination:

• An ultimate free end or termination of a Vein let was called Vein let

termination and the number of the same per square mm of leaf surface was

termed as Vein let termination number.

• It was determined as per Vein let number and can be estimated at same time. It

can be used as a distinguishing character for the leaf of the same species or

different ones, more particularly when the Vein Islet numbers does not give

distinguishing.

• The leaf pieces were taken on to a watch glass and one each was mounted on a

slide in chloral hydrate solution with lower surface of the leaf facing upwards

so that the Veins which are more prominent in the lower surface are seen

clearly under the microscope.

• Usually for this study 6x eye piece and low power objectives were used. The

stage micrometer was to be focused ( 1 mm) and the camera lucida was to be

fixed in such a way that the aperture of it was in the same line with that of the

eye piece.

• Black drawing sheet was placed on the same side of the microscope where

camera lucida was fixed.

• Using a white pencil the first and last of the stage micrometer was removed

and slide was mounted with the leaf specimen and focus in the same way.

• The square drawn in the paper is adjusted in such a way that it lies exactly in

the middle of the field of vision and the image of the leaf piece mounted

appears to be superimposed on the square of the drawing sheet.

80

OBSERVATIONS AND RESULTS

• Starting from one side all the Vein Islet inside the square as well as on the

boundary is to be traced. The Vein let termination within the square only was

taken in to account. To get the exact values it was necessary to take reading

from 4 such squares and trace the Vein Islet within it.

• The Value obtained from the Vein Islet and Vein let termination was

calculated as an average.

3) Observation of Stomatal Index:

• The stomatal number and the Stomatal Index was a very specific criteria for

the identification and characterization of Kakamachi.

• Stomatal number was the average number of stomata per mm2 of epidermis

and the number on each surface of leaf.

• Each stomata consists of two guard cells and the spore was counted as a single

unit. Though this has significance in determining the quality of crude drugs,

this number varies unfortunately, depending on the environmental condition

and geographical sources where the plants were grown.

• Stomatal Index was one of the more distinguishing characteristics for herbal

leafy drugs. It was the percentage praportion of stomata on one side and

epidermal cells plus stomata on the other side.

• In other words, Stomatal Index is defined as the percentage of stomata from

the total number of epidermal cells, which can be explained as;

Stomatal index = S X 10

E + S

S = the number of stomata in a given area of a leaf

E = total number of epidermal cells including trichomes in the area

of leaf.

81

OBSERVATIONS AND RESULTS

• A peeling can be easily taken by partially cutting one of the Veins with a blade

on the lower epidermal region and pulling it. The membranous layer, which

comes out along with the Vein was taken on a glass slide by cutting the thick

Vein section attached to it.

• A few drops of chloral hydrate solution were added and the cover slip was

placed on the top. The peeling was to be sufficiently big enough to cover the

entire field of view when mounted.

• A 10x eyepiece and high powered objective used. 1mm2 of area was taken

with the help of a stage micrometer as described in the previous section.

• On the tracing paper a circle smaller than that of the field of view was drawn

and the camera lucida is fixed in such a way that the circle /square was clearly

visible in the centre of the field of vision.

• For determining the stomatal number it was only to be marked and for each

leaf sample, not lesser than ten determinations should be carried out and the

average index was calculated by using this parameter, Identification and

standerdization were done.

4) Observation of Palisade Ratio:

It can be defined as the average number of palisade cells present beneath each

upper epidermal cell. It can be determined on fine powder. This value remains

constant within a range for a given plant species and was of diagnostic feature for

characterization and identification of Kakamachi.

Like this Stomatal Number, Stomatal Index, Vein Islet Number, Vein

Termination Number, Palisade Ratio, Starch grain Determination.

5) Observation of Extractive Values:

During determination of extractivation following things were observed such as

82

OBSERVATIONS AND RESULTS

Total soluble constitute of the drug in any particular solvent or mixture of

solvents was nothing but the extractive values that was observed and calculated.

The Extraction of Kakamachi with a particular solvent yields a solvent

solution containing different phyto-contituents. The composition of these phyto

constituents in that particular solvent depends upon the nature of the drug and solvent

used. The use of a single solvent can be the means of providing preliminary

information on the quality of a particular drug sample, for example in a drug where

the extraction procedure for the constituents commences with water solvent, any

subsequent aqueous as the extraction on the re-dried residue will give a very low yield

of soluble matter.

6) Observation of Water Soluble Extractive:

During the determination of water soluble extractive following things were

observed such as

• Accurately weighed about 5 gm of powder sample was taken in a conical

flask.

• Correctly added 100 ml of water to it.

• Shaken and kept over night

• Next day observed it and filtered. 20 ml of filtrate was taken in a previously

dried and weighed porcelain dish and evaporated on a hot water bath and

carefully observed.

• Later on weight of the residue was observed which was the water-soluble

extraction.

• After that, observe the percentage and calculated on the basis of air dried

sample.

83

OBSERVATIONS AND RESULTS

7) Observation of pH Value:

A potential is developed across the glass of electrode because of the difference

in hydrogen activity in the solution on the two sides of the glass. The glass electrodes

behave like a concentration cell and so oxidizing and reducing agents do not disturb it

as it is an oxidation reduction cell. The potential of the glass electrode is proportional

to the pH of the solution in which it is immersed.

Connected a power pack of 230 V to the pH meter. Insert the combined

electrode in the socket and adjust the reading of pH and temperature at 30 C, switch

on the instrument. The instrument is filled with distilled water and warmed it. Dip the

electrode in a standard solution of pH and set temperature and the take the reading.

Remove buffers wash and wipe the electrode and dip it in the unknown solution and

take the reading.

84

OBSERVATIONS AND RESULTS

Table No:12

B) Phytochemical observations

Time Acidic Media pH Alkaline Media pH

0 Hours 3.30 8.78

1 Hours 3.35 8.66

2 Hours 3.40 8.31

3 Hours 3.42 8.30

4 Hours 3.49 8.24

5 Hours 3.50 7.87

6 Hours 2.90 7.80

7 Hours 2.91 7.57

8 Hours 2.78 7.34

9 Hours 2.82 7.20

10 Hours 2.83 7.22

11Hours 2.80 7.06

12 Hours 2.91 7.09

1) Observation on Choice of solvent for extraction:

Leaves coarse powder of Solanum nigrum was taken in different solvents such

as petroleum ether, Butenol, Etyl acetate, Chloroform, Ethyl alcohol etc.. put on to the

filter paper directly in different funnels according to the different solvents after seeing

the residue, where residue was minimum that has solubility of the drug in that solvent

is maximum i.e. solubility of Solanum nigrum in ethyl alcohol.

85

OBSERVATIONS AND RESULTS

2) Observation of Extraction:

During extraction the following were observed

• By appropriate technique the coarse powder of Kakamachi is put in the round

fold of filter paper in Soxhlet apparatus so that it can not obstruct any path

ways of Soxhlet apparatus by thermostat mantle where uniform temperature is

maintained.

• During each batch, the cycles were continued till up to extractive factors of the

leaves were get completely extracted in to the solvent then and then only every

batch was stopped. Observation was done so that with this complete extractive

factors were extracted in. After extraction solvents were distilled off,

observation was done so that the solvent is completely distilled off from the

total extraction.

• Semi solid extraction taken off and put over the magnetic stirrer for

concentration of extraction should not be so liquid or completely dried.

3) Observations of Preliminary Phytochemical Test:

1) Test for Sterols:

a) Salkowiski's test

Extract + Conc H2 SO4 + chloroform Solution Shaken Lower layer turns red.

(Red P.P.T) Indicates the presence of Sterols.

b) Libermann –Burschardt test:

Extract + Conc H2 SO4 added Brown ring does not

Chloroform solution + at the sides of test tube shows the junction

Acetic anhydride + and upper layer

Indicates the absence of Sterols dose not turns green

86

OBSERVATIONS AND RESULTS

c) Sulphur test::

Extract + Sulphur Powder Shake downward

Indicates the presence of Sterols

2) Test for Proteins:

a) Extract + Soda lime+ Ammonia Gas is evolved

Test solution

Indicates the presence of Proteins.

b) Test solution + Million's reagent heated does not show white PPT

does not become red.

Indicate Absence of Proteins

c) Test Solution + 1 ml Mercuric Sulphate boil gently does not

10 % H2 SO4 1 % sodium nitrate show red

PPT

Indicates the Absence of Proteins.

d) Biuret test:

Test Soln + 1 ml 5% NaOH heat does not show Violet or pink PPT

+ 2 drops of 1% CuSO4 soln.

Indicates Absence of Proteins.

e) Test soln +Few drops 10% tannic acid heat does not show Violet

or White P.P.T

Indicates Absence of Proteins.

3) Test for Triterpenoids:

a) Salkwoski's test:

Extraction+Conc H2 SO4+chloroform soln Shaken lower layer

allow to stand does not turns red

Indicates Absence of Triterpenoid.

87

OBSERVATIONS AND RESULTS

b) Libermann –Burschardt test:

Chloroform soln + Extract Conc H2 SO4 lower layer doesn' t

+ Acetic anhydride allow to stand turns red.

Indicates Absence of Triterpenoid

c) Tschegajew test:

Extract test soln.+ Excess of water bath No eosin red colour forms.

acetyl chloride and pinch of

zinc chloride.

Indicates Absence of Triterpenoid

4) Test for Alkaloids:

a) Mayer's test:

Test soln + Mayer's reagent Potassium mercuric Observed Grey coloured

iodide precipitate.

Indicates the presence of Alkaloids

b) Wagner's test:

Acidic soln + Wagner's reagent potassium iodide Gives brown

of sample precipitate

Indicates the presence of Alkaloids.

c) Hager's test:

Acid soln + Hager's reagent picric acid observed yellow

of sample saturated precipitate

Indicates the presence of Alkaloids

88

OBSERVATIONS AND RESULTS

d) Dragendroff's test:

Acid soln + Drgendroff's potassium bismuth iodide Reddish brown

of sample reagent precipitate

Indicates the presence of Alkaloids.

5) Test for Carbohydrates:

a) Molisch test:

Test soln +Molisch reagent conc H2 SO4 No purple ring at the junction

Indicates the absence of Carbohydrate.

b) Barford's test:

Test soln + Barford's reagent boiling water bath No brick red PPT

Indicates the absence of Carbohydrate.

c) Benedict's test:

Sample soln + Benedict's reagent boil water bath No reddish brown PPT

Indicates the absence of Carbohydrate

6) Test for Saponin's:

a) Foam test:

Extract+Saponin Shake H2O Formation of stable froth for one minute.

Indicate the presence of Saponin.

b) Heamolysis test:

18%Nacl+disH2O + drop of Blood No haemolysis

18% Nacl+ filterate+drop of Blood under microscope No haemolysis

Indicates absence of Saponin.

89

OBSERVATIONS AND RESULTS

7) Test for Flavonoids:

a) Ferric Chloride test

Alcoholic soln +Few drops of Ferric ( Fecl2) Blackish red colour PPT

chloride

Indicates presence of Flavonoid.

b) Schinoda test:

Alcoholic soln + conc HCl show pink or magneta red

few fragments of magnesium colour

ribbon + conc HCL

Indicates presence of Flavonoid.

c) Zinc HCL reduction test:

Alcoholic soln + Zinc few dropd of HCL No magneta red colour.

Indicates Absence of Flavonoid.

d) Alkaline reagent test:

Alcoholic soln Treated with dilute Yellow colour dilute acid

NaoH colourless Indicates presence of Flavonoid.

e) Lead acetate test:

Alcohol solution few drops of lead Yellow PPT.

acetate 10%

Indicates presence of Flavonoid.

f) Bromine water test: Extraction + Bromine water dissolved Yellow precipitate

Indicates presence of Flavonoid.

90

OBSERVATIONS AND RESULTS

8) Test for Tannins:

a) 1 ml alcoholic soln treated with Fecl2 dark precipitate

Indicates presence of Tannin.

b) Alcoholic Soln treated with lead acetate white precipitate

Indicates presence of Tannin.

c) Alcoholic Solution treated with Bromine water Yellow colour.

Indicates presence of tannins.

OBSERVATIONS OF PRELIMNARY PHYTOCHEMICAL TESTS

Table No 13

Tests Observations

1) Tests for Sterols:

a) Salkowiski's test

b) Libermann-Burchardt test

c) Sulphur test

Turns Red Colour

No Brown ring at junction

Sinks down words

2) Test for Proteins:

a) Extract + Sodalime + Test soln.

b) Test soln + Million's reagent

c) Test soln + Mercuric suphur

d) Biuret test

e) Test soln + 10% Tannic acid

Ammonia gas was evolved

No Red precipitate

No Red precipitate

No violet precipitate seen

No white precipitate seen

3) Test for Triterpenoids:

a) Salkowiski's test

b) Libermann-Burchardt test

c) Tschegajew test.

Lower not turns red

Upper layer turns green

No eosin red colour formed.

4) Test for Alkaloids:

91

OBSERVATIONS AND RESULTS

a) Mayer's test

b) Wagner's test

c) Hager's test

d) Dragendroff.s test

Grey coloured precipitate

Gives Brown precipitate

Yellow precipitate

Reddish Brown precipitate

5) Test for Carbohydrate:

a) Molisch's test

b) Barford's test

c) Benedict's test

No purple ring at junction of two liquids.

No Brick red precipitate

No Reddish Brown precipitate

6) Test for Saponins:

a) Foam test

b) Haemolysis test

Formation of froth

No Haemolysis

7) Test for Falavonoids:

a) Ferric Chloride test

b) Schinoda test

c) Zinc-Hcl reduction test

d) Alkaline reagent test

e) Lead acetate test

f) bromine water test

Blackish red colour precipitate

Pink or Magneta red colour

No Magneta red colour

Colourless

Yellow precipitate

Yellow precipitate

8) Test for Tannins:

a) Solution + Fecl2

b) Solution + Lead acetate

c) Solution + Bromine water

Dark precipitate

White precipitate

Yellow precipitate

92

OBSERVATIONS AND RESULTS

4) Identification by TLC:

About three to six centimeter solvent front migration is sufficient to effect

proper separation Wattman TLC plates produced from 4-5 micro-meter, Silica gel,

with an inert binder to form 200 mm layers about 7 cm development distance was

achieved sample preparation in TLC needs a concentrated solution as very less

amount to be applied and it had been developed using the technique of TLC.

The details of TLC as follows

Table No 14

Plate Size : 20 x 8 cm

Technique : One way ascending

Temperature : 30 C

Examination : Day light after spraying

Plate thickness : 3 mm

Activation temperature : 110 C

Time : 30 min- 1 hr

Detecting Spraying Reagent : Dragendroff's reagent

Absorbent Layer : Silica gel G(Activated) percolated plates

Solvent System : Chloroform

• The Silica gel powder mixed with water and make thin paste ,then with the

help of glass slide the silica was spread on glass plate uniformly.

• After some times the air dried plates are kept in a hot oven at 110 C -120 C

heat was given continuously.

93

OBSERVATIONS AND RESULTS

• For one hour then the prepared sample was kept on one of the plate then

immersed in a solvent up to 10 minutes and then Dragendroff's solution is

sprayed on the plates.

During this procedure following observations were observed:

• The Silica gel slurry taken on plates with the help of glass slide, the silica gel

was spread on glass plates uniformly.

• After sometimes the air dried plates were kept in hot oven at 110 C- 120 C.

heat was given continuously.

• After that, the prepared sample was kept inside the developing chamber with

the plate, then immersed in a solvents up to 30 minutes , closing the plate with

the lid.

• After Dragendroff's reagent was sprayed on the plates.

• Black spot was observed on TLC plate.

5) Observation of U V and I R:

During determination of partial characterization by UV and IR spectroscopy,

the following observations were observed.

• Absorption were measured at a wave length that were generally a

characteristic of the chemical composition of a dissolved absorbing substance.

• Radiant allergy waves range from 200 mm to about 380 mm in the U V

region and from 380 to around 780 mm in the visible region.

• The U V or Visible spectrum of a molecule was the result of change in energy

of a molecule shown or rather than that of a particular bend.

94

OBSERVATIONS AND RESULTS

• The U V visible spectrum of a substance generally not have a high specific

degree but were suitable for quantitative essays for many substances and

useful in additional means of identification.

• The U V spectral analysis was selected as one of the parameter.

• The U V divisible spectra of the sample were recorded in a schimaduae double

beam U V visible recording spectro-photoments ( Model UV-160 A)

• Method extract of the samples after suitable dialatation, were scanned through

200-800 mm and the spectra was recorded.

c) Observation of Experimental Study:

Observation on Selection of Animals:

• The handling of the selected laboratory animals involves two most imporatant

responsibilities on the part of the experiment.

• First, the animal was handled with utmost care so that it does not suffer pain

and secondly, due regard was paid towards the health and well being of the

animal colony.

• Even when they were killed at the end of the experiment, INSA provides the

following observations to followed by all research scholars engaged in the

animal experimentations.

1) Housing with breeding and maintenance of experimental animals to keep them in

physical comfort and good health and to permit them to grow and behave normally.

2) Sources of experimental animals of known genetic health and nutritional status.

3) Observation about technicians and other supportive staff with animals and their use

in experiments.

95

OBSERVATIONS AND RESULTS

2) Sample size:

Albino rat was one of the commonest animals suitable for experimental work

because of its size and sensitivity to most of the drugs. It was also the most

standardized of all laboratory animals. It can be bred to obtain pure and uniform

strains and was found to be very useful in the studies which withstand long periods of

experiments. Hence 24 Albino rats were selected for the study.

I. Inclusive Criteria:

i) Adult healthy Albino rats.

ii) Male Albino rats weighing 110-180 gms.

iii) Albino rats between 90-120 days old were included.

II. Exclusive Criteria:

i) Unhealthy Albino rats.

ii) Weigh range between 110 gms above 180 gms.

iii) Female Albino rats.

iv) Albino rats of below 90 days and above 120 days were excluded.

3) Observation on preparation of the test drug:

Group I –Standard Group: Readily available Ibuprofen suspension was taken and

given to the first group of rats from infant feeding tube and syringe in the dose of 162

mg/kg, The Carragrinine was induced to rats and the inflammation was recorded.

Group II- Trial Group ( Minimum dose)

During the preparation of test to the Group II it was observed that the weighed

extractive fraction was diluted with 1 ml of concentration of that of 1 ml of test drug

is given to the second group of rats from infant feeding tube and syringe through oral

route, after six hours the carragrinine was induced to the rats and inflammation was

recorded, before and after administering the prepared test drug.

96

OBSERVATIONS AND RESULTS

Sample drug = 270 mg/kg.

Group III- Trial Group (Maximum dose)

During the preparation of test drug to the Group III it was observed that the

weighed extractive fraction was diluted with 1 ml of concentration of that of 1 ml test

drug was given to the second group of rats from infant feeding tube and syringe

through oral route, after six hours the carraginine was induced to rats and

inflammation was recorded before and after administering the prepared test drug.

Sample drug= 540 mg/kg.

Group IV (Normal Saline)

1% of normal is prepared with 100 ml of water and 1 ml was taken for the

observation purpose. This was fed orally to this group of rats in a single dose. After

Six hours the already carraginine induced rats. The inflammation was recorded before

and after administering normal saline.

Normal Saline= 0.2 ml in 200 gms rat.

4) Grouping: Table No : 15

Group I Standard Durg Ibuprofen 06 Rats

Group II Trial drug sample Extraction of

Kakamachi(Minimum dose-270

mg/kg)

06 Rats

Group III Trial drug sample Extraction of Kakamachi

(Minimum dose-540 mg/kg)

06 Rats

Group IV Vehicle Normal Saline 06 Rats

97

OBSERVATIONS AND RESULTS

5) Induction Method:

i) For all the 24 Rats, which were grouped into four groups of six each, the

carraginine taken with respective dose at 2 ml disposable syringe with needle was

administered through hind paw of rat without disturbing the normal behaviour.

ii) Initial inflammation was recorded.

iii) Immediately just after the injection of carraginine, inflammation was recorded.

6) Drug Administration:

i) Simultaneously, the prepared test drug sample of Kakamachi was administered

to the Albino rats orally.

ii) At the same time the prepared standard trial drug and 1% normal saline was

administered to Group I, Group II, Group III and Group IV orally respectively.

iii) Three hours after administration of drugs again the inflammation was recorded

in all the four groups by plathysmograph. The variation of inflammation

between two groups was observed.

7) Observation of selected dose:

Trial drug minimum dose -270 mg/kg

Trial drug maximum dose- 540 mg/kg

Normal saline -0.2 ml/ 200 gm body weight

Carraginine- 150 mg /kg body weight. i.e. 30 mg / ml yeast.

Conversion of dose / kg body weight.

Animal dose – Human dose x 0.0118 for 200 gm.

Minimum dose- 3 gm x 0.018= 0.054 gm = 54 mg = 54 x 5 = 270 mg/kg

Trial drug to Albino rats = 270 mg/kg

Maximum dose = 6 gm x 0.018 = 0.108 gm =108 mg =108 x 5 =540 mg/kg

Trial drug to Albino rats = 540 mg/kg.

98

OBSERVATIONS AND RESULTS

8. Duration of Treatment:

i) The first three days observed for the natural behaviours with suitable housing.

ii) 4th day the human –animal infection was observed.

iii) 5th and 6th day kept for 48 hours starvation under observational study.

iv) observed for the increased and decreased conditions of inflammation by

recording.

1) Observation of Anti-inflammatory activity:

i) The Rats were held in a good position where the normal physiological

functions should not affected.

ii) The hind paw of the rat should be in the protruding position.

iii) The hind–paw should be immersed in a 'Y' shape tube of plathysmograph.

iv) Carrageen induced hind-paw after immersing, the mercury level of 'Y' shape

tube suddenly raised .

v) The raised mercury level was measured in mm.

vi) That is the inflammation = raised mercury from 'Y' shape tube in mm.

vii) For group I treated with standard drug after induction of carraginine, reduction

of inflammation was calculated.

viii) For group II treated with trial drug ( Minimum dose ) after induction of

carraginine reduction of inflammation was calculated.

ix) For group III treated with trial drug ( Maximum dose ) after induction of

carraginine, reduction of inflammation was calculated.

x) For group II treated with 1% normal Saline after induction of carraginine the

decreased inflammation was calculated.

xi) At last which group was more potent and more significant that is also

calculated.

99

OBSERVATIONS AND RESULTS

RESULTS OF PHARMACOGNOSTICAL STUDY

1) Results of Morphological Study

Table No :16

Colour Green

Taste Bitter

Size 2.5-9 cm

Shape Ovate

Odour Characteristics acceptable

Touch Smooth

2) Results of Microscopical Study:

By the section method and by staining process, the following results are seen

under the microscope.

Trachoma, Parenchyma cells, Xylem, Phloem, vascular Bundles, Stomatal

cells, Palisade cells, Starch grains, Lower epidermis and Upper epidermis. While

considering the cytomorphological aspects, in the case of whole drug the cell

distribution can be determined by sectioning and in powders some degree of cellular

aggregations and organization is retained. To evaluate all the cell parameters, the

distribution of the basic cell types as well as cell inclusion has to be studied. The basic

types include cellular parameters of plant cells as follows;

100

OBSERVATIONS AND RESULTS

Parameters of plant cells

Table No: 17

Physical evaluation:

Sl No Microscopical cells Values

1 Trachoma 15-20/mm2

2 Parenchyma cells 30-35/mm2

3 Xylem 20-25/mm2

4 Phloem 20-25/mm2

5 Vascular Bundles 10-15 in bundles

6 Stomatal cells 20-25/mm2

7 Palisade cells 15-20/mm2

8 Starch grains 20-25/mm2

9 Lower epidermal cells 20-25/mm2

10 Upper epidermal cells 20-25/mm2

11 Fibers 30-25/mm2

12 Collenchymal cells 20-25/mm2

The sample of leaves of kakamachi are analyzed by employing various

parameters as mentioned in materials and methods and the data evolved had been

presented here in the tabular form.

101

OBSERVATIONS AND RESULTS

In physical evaluation the following results are seen as;

Table No:18

Sl No Parameter Values

1 Stomatal number 16-25/mm2

2 Stomatal index 18.6 mm

3 Vein islet number 26/mm2

4 Vein termination number 15/mm2

5 Palisade 20-25/mm2

6 Starch grain 40-60/mm2

7 Epidermal 24/mm2

8 Starch grain determination 24/mm2

9 pH value 8

(B) Results of phytochemical study:

1) Choice of solvent:

Table No :19

Sl. No. Solvent Soluble Sparingly soluble Insoluble

1 Distilled water + - -

2 Solvent ether - - +

3 Petroleum ether + + -

4 Acetone - - +

5 Benzene - + -

6 Toluene - - +

7 Chloroform - + -

8 Ethyl alcohol + + -

9 Xylene - - -

10 Carbon tetrachloride - - +

Ethyl alcohol 90%

102

OBSERVATIONS AND RESULTS

(2) Extraction

Table No: 20

Kakamachi of each batch Solvent Extract

Coarse powder 120 gm Ethyl alcohol 650 ml 10 gms

(3) Preliminary phytochemical tests

Table No:21.

Tests Results

(1) Test for sterols

(a) Salkowiski’s test

(b) Liberman’s-Burchard test

(c) Sulphur test

+ve

-ve

+ve

(2) Test for proteins

(a) Extract + soda lime + test solution

(b) Test solution + Million’s reagent

(c) Test solution + mercuric sulphate

(d) Biuret test

(e) Test solution + 10% tannic acid

+ve

-ve

-ve

-ve

-ve

(3) Test for Triterpenoids

(a) Salkowiski test

(b) Liberman’s-Burchard test

(c) Tschegajew test

-ve

+ve

-ve

(4) Test for alkaloids

(a) Mayer’s test

(b) Wagner’s test

+ve

+ve

103

OBSERVATIONS AND RESULTS

(c) Hager’s test

(d) Dragendroff’s test

+ve

+ve

(5) Test for carbohydrate

(a) Molisch test

(b) Bar ford’s test

(c) Benedict’s test

-ve

-ve

-ve

(6) Test for Saponin’s

(a) Foam test

(b) Haemolysis test

+ve

-ve

(7) Test for flavonoids

(a) Ferric chloride test

(b) Schinoda test

(c) Zinc-HCl reduction test

(d) Alkaline reagent test

(e) Lead acetate test

(f) Bromine water test

+ve

+ve

-ve

+ve

+ve

+ve

(8) Test for tannin’s

(a) Solution + FeCl2

(b) Solution + lead acetate

(c) Solution + bromine water

+ve

+ve

+ve

(5) Identification by TLC

* The T.L.C. study of the samples was carried out by using different conditions

to evolve suitable T.L.C. pattern, and the data was observed.

104

OBSERVATIONS AND RESULTS

* When viewed under long U.V radiation showed only one spot at R 0.98 but no

spot was seen in the sample.

* T.L.C of the alcoholic extract on silica get ‘G’ plate using n-Butanol: Acetic

acid-water (4:1:5) shows under UV (366 nm) three fluorescent zones of Rf.

0.54 (bright sky blue) 0.84 (light sky blue) and 0.93 (bright sky blue)

* On exposure to Iodine vapor seven spots appear at Rf

(0.15,0.27,0.54,0.67,0.78 and 0.93) all yellow.

* On spraying with 5% Methanolix-sulphuric acid reagent and heating the plate

at 1050 C for ten minutes, eight spots appear at Rf. 0.15,0.27,0.32,0.38 ( all

grey), 0.54 (yellow) 0.67, 0.84 (light grey) and 0.93 (brown)

* The chromatogram after spraying with Dragendroff’s reagent followed by

heating at 1100 C for 5 minutes several spots at Rf. 0.12 (indistinct), 0.32

(violet), 0.58 ( faint violet) and 0.73 (pinkish violet), 0.80 (faint violet), 0.90

(faint violet), and 0.93 (violet).

* Perusal of the evolved chromatograms clearly shows that the chromatographic

patterns of the sample are quite different in all the conditions indicating wide

difference in the chemical composition of Kakamachi.

* The evolved chromatograms will be very useful for the analysis and

identification of Kakamachi.

5) U.V. Spectrophotometric Analysis:

* The U.V Spectra of Ethyl alcohol extract, after suitable dilution of the samples

were recorded and presented. The spectra of the Kakamachi shows absorption

peak at 210 nm 207nm respectively.

* The comparison of the spectra presented shows difference in indicating

difference in chemical composition in Kakamachi.

105

OBSERVATIONS AND RESULTS

C) Results of Experimental study:

I) Analytical values of inflammatory activity:

Group- I treated with Standard Drug

Table No: 22

Shows the reading for the inflamed hind paw volume before and after the

treatment of Group-I

Sample Initial Paw volume Paw volume after 3 hours Difference

1 1.44 1.24 +0.20

2 1.48 1.46 +0.02

3 1.40 1.30 +0.10

4 1.48 1.20 +0.28

5 1.40 1.30 +0.1

6 1.40 1.30 +0.1

Table no: 23

Showing the statistical data of group – I

SD SE t P

0.091 0.037 13.548 <0.0001

Graph showing the mean value of standard group

0.2

0.02

0.1

0.28

0.1 0.1

0

0.05

0.1

0.15

0.2

0.25

0.3

Diff

eren

ce

1 2 3 4 5 6

Samples

106

OBSERVATIONS AND RESULTS

Group II treated with Trial drug Minimum Dose

Table No: 24

Shows the reading of the inflamed hind paw volume before and after treatment

of Group- II

Sample Initial paw volume Paw volume after 3 hrs Difference

1 1.44 1.38 +0.06

2 1.46 1.39 +0.07

3 1.43 1.38 +0.05

4 1.40 1.34 +0.06

5 1.40 1.30 +0.1

6 1.40 1.36 +0.06

Table No: 25

Showing the statistical data of group II

SD SE t P

0.42 0.017 3.438 <0.02

Graph showing the mean value of Trial group (minimum dose)

0.060.07

0.050.06

0.1

0.06

0

0.02

0.04

0.06

0.08

0.1

0.12

Diff

eren

ce

1 2 3 4 5 6

Samples

107

OBSERVATIONS AND RESULTS

Group III treated with Trial Drug (maximum dose)

Table No: 26

Shows the reading of the inflamed hind paw volume before and after treatment

of Group III

Sample Initial paw volume Paw volume after 3 hrs Difference

1 1.44 1.26 +0.18

2 1.48 1.44 +0.04

3 1.40 1.38 +0.02

4 1.48 1.20 +0.28

5 1.40 1.30 +0.1

6 1.40 1.30 +0.1

Table No: 27

Showing the statistical data of group III

SD SE t P

0.091 0.037 11.548 <0.001

Graph showing the mean value of standard group (maximum dose)

0.18

0.040.02

0.28

0.1 0.1

0

0.05

0.1

0.15

0.2

0.25

0.3

Diff

eren

ce

1 2 3 4 5 6

Samples

108

OBSERVATIONS AND RESULTS

Group IV treated with control – Saline water

Table No:28

Shows the reading of the inflamed hind paw volume before and after treatment

of Group IV

Sample Initial paw volume Paw volume after 3 hrs Difference

1 1.49 1.48 +0.01

2 1.33 1.31 -0.02

3 1.40 1.39 +0.01

4 1.38 1.37 +0.01

5 1.49 1.48 +0.01

6 1.32 1.31 +0.01

Table No: 29

Showing the statistical data of group IV

SD SE t P

0.013 0.005 0.938 >0.10

Graph showing the mean value of control group

0.010.01

-0.02

0.01 0.010.01

-0.025

-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

0.015

1 2 3 4 5 6

Samples

Diff

eren

ce

109

OBSERVATIONS AND RESULTS

STATISTICAL ANALYSIS

The data obtained through the experimental study as shown in the tables being

analyzed statistically based on the student t-test values as follows.

• In standard group the result was highly significant with p value <0.0001

• In the trial group (minimum dose) the result was moderately significant with P

value <0.02

• In the trial group (maximum dose) the result was highly significant with P

value 0.001

• In the control group the result was non significant with P value >0.10

Therefore, it is ascertained that Kakamachi can play a highly significant role

with maximum dose and significant role with minimum dose in reducing

inflammation being equally effective as the standard drug Ibuprofen.

110

DISCUSSION

DISCUSSION

The title of the present study is “Pharmacognostical Studies and Anti-

inflammatory effect of “Kakamachi” on Albino rats”. This protocol includes the

systematic study of the ideal drug Kakamachi, its specific characteristics,

photochemical study for determination of different chemical components in the Trial

drug. Experimental study is done for Anti-inflammatory activity. Hence this title

represents the complete wholesome study of research work.

In Ayurveda, there are so many drugs mentioned under the anti-inflammatory

property. In that, Kakamachi is one, which is a major one. The present study is of

Pharmacognostical, phytochemical and experimental study of Kakamachi, with its

special reference to anti-inflammatory activity. Under this the literature review,

disease review, aims and objects of the Kakamachi are studied, the materials and

methods, observations and results were discussed and concluded.

Shotha, Shopha and Svayathu-the terms used may be different in different

contexts, but all our acharyas has given much importance to this condition. They

have devoted separate chapters for the description of the Nidana Panchakas and

Chikisa. Sushruta, while describing the Nirukti of Sopha “Ekadeshothitha Shopha

Ityuchyate” meaning oedema arising in any one part of the body is Shopha. While,

describing vishesa laxanas of Shopha, he uses the word Shvayathu, which has also

been used during description of sarvasara Shopha. So it may be understood that all

the three terms are synonymous.

To understand the disease entity for experiments based on modern science, an

attempt has been made by putting correlation between Shotha and inflammation. This

is according to the respective explanations of the similarities in their symptoms.

Inflammation is local response of living mammalian tissues elicited as a defense

111

DISCUSSION

reaction in order to eliminate or limit the spread of injurious agents as well as to

remove the consequent necrosed cells and tissues. A series of phenomenon including

increase in the vascular permeability, accumulation of exudates and bringing into

action of many mediators like the prostaglandin etc,. that plays an important role are

set into action.

Till the date a number of studies have been carried out to screen the various

herbs for anti-inflammatory action to put forth the alternative diseases. Herbal

remedy, which could take the place of the synthetic drugs of the new era.

Kakamachi is a drug popularly known for its efficacy in skin disorders and

vruna shodana in various vruna. It has also been highlighted for its efficacy in

Hridaya roga, Rakta Shodaka, Shothahara and Hypertension and also other ailments.

Many of the nighantukaras have mentioned shothahara property of Kakamachi in their

nighantus.

Shweta and Rakta, the two varieties of Kakamachi are seen growing wildly in

the northern part of Karnataka. The drug is Tikta in Rasa, laghu, Tiksana and Virya is

|Ushna and where as Vipaka is Katu. Panchanga of Kakamachi is considered to be its

official part. Solanum nigrum is taken up for the present study. The drug was

identified by botanists and faculty members of PG department of Dravyaguna, before

beginning the study.

The present study includes the morphological, microscopical, physical

evaluations. Under the morphological discussions, the points were discussed such as

materials, drugs useful parts, collection of materials, equipments, chemical, methods

were discussed . Under the discussion of microscopical study the section methods,

staining process, chemical methods were discussed.

112

DISCUSSION

Discussions of Pharamacognostical Study:

Present Pharmacognostical study includes morphological, physical evaluation

of the drug. In the discussion of morphological study, following were observed i.e

taste-Bitter, size, shape etc.

In discussion of microscopical study, following were seen.

The T.S. of leaf shows a non-glandulous trachoma especially on the mid rib

region. The trachoma are of two types i) Very slender, multi-cellular and uniseriate

straight or bend ii) Very smaller with thin cell wall, globose and conical apex, the

vascular bundles are seen at the middle of the midrib region. the vascular bundles are

semi lunar in outline with an upper readily arranged xylem tissue and lower phloem

tissue. It shows outline with margin. The epidermis possesses multi-cellular

enumerous slender non-glandular trachomas. Below the bulge of the epidermis there

are small patches of collenchyma cells and matured tissues. There are parenchyma’s,

cortex and small patches of sclerenchyma fibers are observed and also beneath

sclerenchyma fibers there are distinct vascular bundles ranging from 8 to 10 in

numbers. Below the cortical parenchyma there is a distinct endodermis consists of

tangentially elongated parenchyama cells. The pericycle group of cells which are

distinct and comparatively wide, the phloem tissue is followed by a group of xylem

tissue. The xylem is made up of large vessels and traechids most of parenchyma cells

of cortex and medullury rays contain small prismatic crystals of calcium oxalate and

few starch grains.

By discussion of physical evaluation of the Kakamachi, the coarse powder of

the same is mixed with water and shaken well, after keeping this solution over night is

filtered, the next day and on evaporation minimum solubility of the drug in water was

seen. Heaves coarse powder of the Solnum nigrum was taken in different solvent.

113

DISCUSSION

The solubility of solanum nigrum is maximum in ethyl alcohol was seen. The

extraction of coarse powder of Solanum nigrum was done by using soxhlet apparatus

and uniformally maintaining the temperature by using a thermostat with ethyl alcohol

of 90% as solvent. A magnetic stirrer is used. pH value of the drug was seen.

Discussion of Phytochemical analysis:

Here the phytochemical study performed to identify the active chemical

components such as alkaloid, sterols, carbohydrates, tri-terpinoids and tannins etc.

Before their chemical tests, the coarse powder of Solanum nigrum was

subjected to exhaustive extraction by soxhlet apparatus around 18 hours in different

batches of 90 % ethyl alcohol. The extractive fractions of the component shows

different chemical constituents such as alkaloids with yellow precipitate,

carbohydrates with brick red precipitate, Flavonoids with maganata red or pink

precipitate colour. Sterols with red precipitate. Tritrepenoids with yellow precipitate

saponins with stable froth and tannins with white precipitate indicates presence of

active components.

In Ayruveda the medicinal values of the plants are mainly attributed to active

chemical components such as sterols, carbohydrates triterpinoids and alkaloids,

tannins etc.

In the present study, all the phytochemical components of the Solanum nigrum

were tested qualitatively by improving specific chemical tests. Before these chemical

tests, the air dried seeds of Solanum nigrum were subjected to exhaustive extraction

by Soxhlet apparatus around 18 hours with different batches with ethyl alcohol of

90%. Which was the prime aim of the present protocol.

The discussion of phytochemical analysis gives following findings such as

114

DISCUSSION

Discussion of UV and IR spectrum:

Under the discussion of the physical evaluation, different things were

discussed, what are the different materials are used which drug was used, what

different equipment is used which chemicals were used the methods which are to be

followed were discussed successfully. The methods for Vein Islet, Vein let

termination are discussed and also for the Stomatal Index, Palisade Ratio, the suitable

methods are discussed.

Discussion on Identification by TLC method :

By following the appropriate methodology T.L.C preparation Black spot was

observed on TLC plate. This spot indicates the active chemical components present

inside the diluted solutions. It shows following findings as at 0.54 bright sky blue

colour. On exposure of Iodine vapour, it shows seven yellow colored spots. On

spraying with 5% methonolic-sulphuric acid reagent and heating plated at 1050 C for

thirty minutes shows eight spots appears at Rf. Such 0.15, k 0.27, 0.32, 0.38 with

showing green colour. Like this different findings were discussed.

The 20x8 cm plates are taken. Silica gel is the most commonly used absorbent

and is evenly on these plate as slurry in water in a 250 mm thick layer by means of a

mechanical spreader or manually. The plates are then air dried and then activated at

100 to 105 0 C for 30 to 45 minutes. Usually the solvent is allowed to ascend to a

distance of 10 cm from the base line and therefore it is quite convenient to draw a

straight line at this level by means of a mounted needle. The layer of stationary phase

has been rendered discontinuous, a Small amount of the sample in solution is applied

to the chromate plate using a template. Spot applications must be as small as possible

and it is best done by means of a capillary tube.

115

DISCUSSION

As the component of the mixture moves up the plate, it tends to diffuse and

thus the size becomes increasingly larger. If therefore, the initial spot is larger, than

the components which separate only slightly will merge together, and thus the

resolution will not be discernable. The abbreviations indicating the particular

substance applied are scratched with a mounted needle on the adsorbent layer well

above the solvent front line at 10 cm level. The abbreviations must never be scratched

on the absorbent surface below the base line and below any solvent front line drawn.

This will disturb the even flow of the solvent render the plate useless.

The plate is then placed in the chroma tank. The solvent is now allowed to

ascent up the plate until H mark reaches the 10 cm line. Note the room temperature

carefully because this is an important consideration. The plate is then removed from

the tank dries with hair drier and examined first in day light and then finally sprayed

with suitable Dargendroff’s reagent.

Any spots observed under these conditions are outlined with a mounted

needles. The line as we see is extremely important in calculating the chromatographic

parameter. The Colour of each spot after spraying is noted. When the examination is

complete, a copy of the TLC plate is made by means of a sheet of tracing paper

carefully noting the spot abbreviations base line and solvent front.

It is important to note the colours and the manner and conditions in which they

appear, as also other relevant particulars. The information should be recorded in the

tracings.

Discussion of UV and IR:

Different chemicals when subjected for photometers in white light (including

UV) have specific affinity to absorb or to transmit a particular ranges of wavelength

which relates to that compound, speaks metric analysis which involves the

116

DISCUSSION

measurement of the ability of the dissolved solutes to absorb light of definite and

arrow wavelength ranges, these absorption are measured at a wavelength that are

generally a characteristic of the chemical composition of a dissolved absorbing

substance. Radiant energy waves range from 200 nm to about 380 nm in the UV

region and from 380 to round 780 nm in the visible region. The UV or visible

spectrum of a molecule is the result of change in energy of a molecule as a show or

rather than of a particular bend, the UV and visible spectras of a substance generally

do not have a high degree of specific, but they are suitable for quantitative assays for

many substances and useful as additional means of identification. Hence, the UV

spectral analysis was selected as one of the parameter. The UV, visible spectra of the

sample was recorded double beam UV visible recording spectrophotometer (Model

UV-160 A)

The IR spectrum of the alkaloid was recorded on Perkins elumes model 183 at

medium scan speed by applying KBr disc. It involves the measurement of the ability

of the dissolved solutes to absorb light of definite and narrow wavelength ranges.

Radiant energy waves range from200 nm to above 380 nm in the UV region from 380

nm to around 780 nm in the visible region. The U.V and visible spectrum of a

substance generally do not have a high degree of specific radiation but they are

suitable for quantitative assay for many substances and useful in identification.

The spectrum of powder of Solanum nigrum was seen. Hence the U.V spectral

analysis was selected as one of the parameter.

Discussion on experimental study:The design of the study on inflammation was made

on animal experimentation. Albino rats weighing between 110-180 gms were selected

carefully for the evaluation. Four groups of animals were selected for the studies

based on the following pattern.

117

DISCUSSION

Group I : Standard Group : Ibuprofen suspension was purchased and fed orally to the

albino rats at the dosage of 162 mg/kg.

Group II : Trial Group – The Solanum nigrum linn extracted was weighed and given

to trial group in minimum dose to the albino rats at the dosage of 270 mg/kg.

Group III : Trial Group – The Solanum nigrum linn extracted was weighed and given

to trial group in maximum dose to the albino rats at the dosage of 540 mg/kg.

Group IV : Control Group – 1% Normal saline was fed orally to the albino rats at the

dosage of 1ml each animal in the single dose.

Carraginine (0.1 ml of 1% w/v in saline) was used to induce inflammation in the left

hind paw of all the rats keeping the right paw as control. The paw volume of the rats

of four groups, before and after injecting Carraginine was measured using

Plethismograph. Paw volume was measured hourly for three hours after injecting the

Carraginine and observations done to see the difference in paw oedema.

Inflammation was observed between 15-30 minutes after induction of

Carraginine. On the analysis of observations it is found the albino rats in the control

group did not show any improvement by three hours. But the rats in the trial group

with minimum dose have shown good response after three hours, where as trial group

with maximum dose and standard drug showed improvement during second and third

hour of injecting carraginine.

Statistical analysis showed that the result in the trial group with maximum

dose is almost nearer to that of standard group.

Development of oedema induced by carraginine is commonly correlated with

the early exudative stage of inflammation, one of the important processes of

inflammatory pathology. In the beginning of carraginine injection, there is sudden

elevation of paw volume in relation with histamine mediators. After one hour, the

118

DISCUSSION

inflammation is increased gradually and was elevated during the later three hours.

This second phase could be due to the liberation of prosto glandin and kinins, which

accompanies leucocytes migration.

Animal experimentation has its limitation in the evaluation of inflammation.

All the symptoms cannot be evaluated as done in clinical study. Here only the utseda

or tumor can be evaluated through the aid of plethismography by which the reduction

of inflammation can be calculated.

All the statistical data and graphs are presented in the tables.

Thus through animal experimentation, it is found that kakamachi has highly

significant action as anti-inflammatory drug with maximum dose, where as

moderately significant action with minimum dose.

The samprapti of shopha begins by vitiation of kapha, rakta and pitta, which

enter the bahya siras and in turn vitiates the vata located there. Thus sroto rodha is

caused which spreads to the areas in the viscinity and shopha results.

The drug is tikta in rasa, laghu in guna ushna veerya and katu in vipaka. Ushna

veerya is known to act on vata and kapha and tikta rasa reduces pitta and cleans the

rakta. Laghu guna also act antagonistically on kapha. Katu in the drug contracts

organic tissues and lessens its secretions and in practice it is found that the katu effect,

some times tend to overlap with anti inflammatory activity to some extent.

Thus the Gunas of trial act all together upon pitta, rakta, kapha during

samprapti vighatana and eventually pacifies these. Once the vitiated pitta, rakta, and

kapha are brought to the normal state the vayu gets pacified thus clearing the

srotorodha relieving shotha.

119

CONCLUSION

CONCLUSION

1) Review of classical and modern literatures shows that the trial drug Kakamachi

is having significant Shothahara property.

2) Shotha manifests as disease itself and also as associated symptom.

3) Comparing the results from the control, trial groups especially makes out

inhibitor activities of Kakamachi in inflammation.

4) Statistically trial drug with maximum dose and standard drug are equipotent.

5) Thus, through experimental studies, the trial drug has significant anti

inflammatory action in Albino rats.

6) No adverse effects were seen and well tolerated by Experimental animals.

120

RECOMMONDATIONS FOR FUTURE STUDY

RECOMMONDATIONS FOR FUTURE STUDY

1. To get scientifically based description of Histopathology of cells or tissues,

pharmacological study is necessary.

2. To assess the claim made on Kakamachi in regarding inflammation, clinical study

is necessary in all phases.

121

SUMMARY

SUMMARY

The present dissertation entitled “Pharmacognostical studies and anti-

Inflammatory effect of “Kakamachi” on albino rats”. It contains following parts such

as-

I) Introduction.

II) Reviews of Literature.

III) Pharmacognostical study.

IV) Phytochemical study.

V) Experimental study.

VI) Discussion.

In the first part a brief introduction was given which deals with the importance

of plant, number of the plants in the Brihatrai and Nighantus in different Vedas,

Samhita kala, Siddha, Unani and adhunik kala with its some reference for shothahara

activity. Aim of selection of the plant kakamachi and the anti-inflammatory activity.

The aim and objectives. Material and methods and plan of the study are given.

In the second part comprises of Review of literature which is divided into two

section viz. Drug review and Disease references in different Nighantus, Samhitas,

Veda and in different modern books along with its different synonyms, Properties

Doshaghnata, Roghaghnata and also the Botanical description of plants such as

vegetative characteristics, floral characteristics, floral formula, floral diagram etc and

its family description with its medicinal value and chemical composition. And its

Amayika prayoga.

122

SUMMARY

Next section contains disease review. In this chapter, description of

inflammation with its introduction, definition and terminology, causes of

inflammation, classification of inflammation, General mechanism of inflammation,

Complication of instantiation, Proliferative tissue changes in inflammation, Review

on anti-inflammation, Classification of anti-inflammation, Management of

inflammation and action of Non-steroidal anti-inflammatory drugs.

Third part deals with pharmacognostical study of the leaf which includes

macro morphological studies, microscopic studies and physical evaluation of the

plant leaves.

This part of the study deals with materials and methods which is the integral

part, which includes material and methods for pharmacognostical study

phytochemical study and experimental study. The aim of the materials assessment is

to evaluate the morphological, microscopical and physical evaluation of the Solanum

nigrum. Coarse powder of Leaves of solanum nigrum, sense organs, microscope,

chemical test tubes, filter paper, pipette and weighing box, beakers, soxhlate

apparatus, redistilled water condenser, conical flask, burner, ethyl alcohol, different

test chemicals, T.L.C kit, hot oven, watt mann glass plate, sprayer, spectroscope,

KBr.disc, Albino rats etc. Were some of the materials used for present study.

To evaluate the effects of Solanum nigrum method, staining process method

for identifying and to know the physical properties of chemical compound, physical

method, extraction methods, solvent selection procedure, different preliminary

photochemical tests, identification by T.L.C method and characterization by

spectroscopy method, animal selection, dose fixation, grouping method are

summarized in this part.

123

SUMMARY

The pharmacognostical study of coarse powder of kakamachi. It includes the

macroscopic and microscopic study, which helps for evaluation of characteristics and

identification of Solanum nigrum with its nature, colour, taste, size, shape and

description of physical constants of the present drug to evaluate their different values.

The phytochemical study of this part includes choice of solvents for extraction

with different solvents like ethyl alcohol, petroleum either, ethanol, chloroform and

water to known the chemical constituents in the drug. Extraction of coarse powder of

Solanum nigrum, with soxhlate apparatus in different batches and the extraction

process was carried out for about 18 hours. The extraction subjected to re-distillation

for recovery of solvents and to obtain a semi-solid extract and concentrated on

magnetic stirrer the weight of each batch was recorded in detailed examination.

The sample drug was subjected to the T.L.C in which the separation takes

place in short time and better resolution and sensitivity can be obtained with smaller

size which is very useful in qualitative and quantitative analysis of compounds are

summarized. The alkaloid fraction was subjected to spectral analysis, with different

chemicals. When subjected for photometry in UV light, which is having specific

affinity to absorb or to transmit a particular range of wave length which is related to

that compound are broadly explained in this part of study.

Experimental study deals with animal experiments and anti-inflammatory

activity. Here the efficacy of Solanum nigrum by its extraction was evaluated against

inflammation. Selection criteria of animals, preparation of test drug with divided

doses on 24 albino rats, grouping method of induction of carraginine. Route of

administration of drug with respective doses of it etc.

124

SUMMARY

Determination of anti-inflammatory activity evaluated on albino rats

maintaining all the procedures of selection, administration dose, route of

administration. Carraginine induction method, duration of treatment etc. description

given in that part. Systematically procedure was done and description is given in that

part of study.

Thus by observing and analyzing the results obtained during this study a

conclusion was drawn.

125

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34) Bertan G. Katzung M.D. Ph.D Basic and Clinical Pharmacology 7th edition 1998

U.S.A:229.

35) Frenchs Index of Differential Diagnosis 13th edition Edited by Ian A.D. Bouchier

Harola Ellis and peter R. Fleming M.D. F.R.C.P London U.K. Butterworth

Heinemann. Publication:335.

36) A Textbook of comparative General Pathology mechanisms of Disease 2nd edition

David o slauson Bary J. cooper maja M. suter Ithaca New york:210.

37) Pathophysiology principles of Disease Martha. J. Miller. W.B. Saunders company

Gaku Shoin- Saunders 1983:540.

38) Robbins Pathologic Basis of Disease 6th edition Ramzi. S. Cotroan M.D Vinaykumar

and Tucker collins Boston saunders An imprint of Elsevier;501-519.

39) General and systematic pathology edited by J.C.E Underwood. 2nd edition. Churchill

living stone Tokyo 1996:221-244.

Journals

40) Indian journals of medical Research.

41) Journal of Indian drugs

42) Journal of Indian Medicine Yoga and Homeopathy

43) Indian Journal of Experimental Biology

44) Ancient Science of life.

45) Indian journal of Pharmacology.

46) Nagarjun

47) Ayur mediline

48) Bheshaj 2005

Websites

49) http:// www herbs. Org

50) http:// www. Google. Com

51) http: // www. Ayurvedca. Com

52) http: // www. Ask. Com