“a comparative antimicrobial study of navasadara …

“A COMPARATIVE ANTIMICROBIAL STUDY OF NAVASADARA SATVA BY TAKING TWO DIFFERENT

SAMPLES OF NAVASADARA”

By

Dr RAGHUVEER B.A.M.S

Dissertation submitted to the Rajiv Gandhi University of Health Sciences, Karnataka Bangalore,

In partial fulfillment Of the requirements for the degree of

AYURVEDA VACHASPATHI

In

RASASHASTRA

Under the Guidance of

Dr. (Smt) P. P. DINDORE M. D.

POST GRADUATE DEPARTMENT OF RASASHASTRA

K.L.E’S SHRI. B.M. KANKANAWADI AYURVEDA MAHAVIDYALAYA, POST-GRADUATE STUDIES CUM

RESEARCH CENTRE, SHAHAPUR, BELGAUM.

2009

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled “A COMPARATIVE

ANTIMICROBIAL STUDY OF NAVASADARA SATVA BY

TAKING TWO DIFFERENT SAMPLES OF NAVASADARA” is a

bonafide and genuine research work carried out by me under the guidance

of Dr. (Smt) P. P. DINDORE, M. D. Professor, Department of Rasashastra

and Bhaishajya Kalpana, Post Graduate Studies cum Research Centre,

K.L.E. Society’s Shri B. M. Kankanawadi Ayurveda Mahavidyalaya,

Shahapur, Belgaum.

Date: DR. RAGHUVEEER P.G. Scholar

Place: Belgaum

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “A COMPARATIVE


TAKING TWO DIFFERENT SAMPLES OF NAVASADARA”

Is a bonafide research work done by Dr RAGHUVEER, Department of

Rasashastra, Post Graduate Studies Cum Research Centre, and K.L.E

Society's Shri. B.M. Kankanawadi Ayurveda Mahavidyalaya, Shahapur,

Belgaum, in partial fulfillment of the requirement for the degree of

AYURVEDA VACHASPATHI.

Date: Guide

Place: Belgaum Dr. (Smt.) P. P. DINDORE M. D. Professor Department of Rasashastra & B K

ENDORSEMENT BY THE HOD, PRINCIPAL OF THE INSTITUTION

This is to certify that the dissertation entitled “A COMPARATIVE


TAKING TWO DIFFERENT SAMPLES OF NAVASADARA”

is a bonafide research work done by DR RAGHUVEER under the

guidance of DR.(smt) P. P. DINDORE M.D .Professor, Department of

Rasashastra and Bhaishajya Kalpana, Post Graduate Studies Cum

Research Centre, K.L.E Society's Shri. B.M. Kankanawadi Ayurveda

Mahavidyalaya, Shahapur, Belgaum.

Dr.R.C.MATHAD. BSAM Dr. B.S.PRASAD Prof & H. O. D. Principal Department of Rasashastra & Post-Graduate Studies Cum-Research Centre, Bhaishajya Kalpana K.L.E. Society’s Shri. B.M.Kankanawadi Ayurveda Mahavidyalaya, Shahapur, Belgaum Date: Date:

Place: Belgaum Place: Belgaum

COPYRIGHT

DECLARATION

I hereby declare that the Rajiv Gandhi University of Health

Sciences, Karnataka shall have the right to preserve, use and disseminate

this dissertation in print or electronic format for academic / research

purpose.

Date: Dr RAGHUVEER P.G. Scholar

Place: Belgaum

ACKNOWLEDGEMENT

I take this opportunity to express my deep sense of gratitude towards

my guide Dr(Smt) P. P. DINDORE M.D. Professor, Department of Rasashastra and

Bhaishajya Kalpana, Post Graduate Studies cum Research Centre, K.L.E. Society's

Shri B.M. Kankanawadi Ayurveda Mahavidyalaya, Belgaum and my co-guide

Dr.C.C.Gavimath Professor, Department of Biotechnology, K.L.E’s Engineering

college, Belgaum for their valuable guidance, critical suggestions, constant

encouragement and overall supervision to complete this dissertation work.

I also express my sincere thanks to Dr.R.C.Mathad BSAM Professor and Head of

the Department Rasashastra and Bhishajya Kalpana and Vice- Principal, for his

constant help during the course of my study and preparation of this dissertation.

I express my thanks to Dr. B.S.PRASAD,M.D, Ph.D Principal, Shri

B.M.Kankanawadi Ayurveda Mahavidyalaya, Belgaum, who has provided all the

necessary facilities for the undertaken study.

I express my thanks to my teachers Dr.R.S.Hiremath, Dr.P.G.Jadar,

Dr.N.M.Hampannavar, Dr.S.M.Patil, for continuous inspiration guidance and

supervision.

My sincere thanks to Dr.M.C.Patil, Dr.G.N.Danappagoudar,

Dr.R.S.Sarashetti, Dr.S.K.Hiremath, Dr Yogesh, Dr.B.B.Joshi., Dr.K Baldaniya,

Dr Samudri, Dr Rudrakshi, for their valuable guidance.

I also express my thanks to Dr. Kishore Bhat M.D., Microbiologist,

Mr Mayur who helped me a lot during Anti microbial study, Dr. Revati of

Bangalore Test House, Mr Mangesh Bhide foundation Pune, Miss Trupti IIT

Mumbai for their cooperation in Analytical part.

I thank Dr Savita Bhosale for her help and sisterly affection. I am greatful

to my senior friends Dr.M.B.Gundakalle, Dr.Nataraj, Dr.Manoj Patil, Dr

Shivaraj, Dr.Prasanna, Dr.Ramesh K, Dr.Manjunath Gavimath, Dr Basavaraj

Ganti, Dr.Archana Joshi , Dr.Poornima B, Dr.Harshita M, Dr Rupa, Dr Vyshali,

Dr Savita Jadhav, Dr Priya, Dr Shubha, Dr Veena , Mr Ajit Lingayat and My

Dear colleagues Dr Surekha, Dr Nitu, Dr Deepti, Dr Mahadev Shinde Dr

Bhagyashree, Dr Amit L ,Dr Katkar, Dr Santosh, Dr Gaurav, Dr Vikas, Dr Mane,

Dr Samir, Dr Deepti Patil, Dr Ashwini, Dr Rabb, and all my junior friends for their

encouragement through out my study and making my stay in belagavi memorable.

It is my great privilege to express my gratitude for my senior friend late.

DR.Shivkumar B, PG Scholar, Dept of Rasashastra Gadag who encouraged and

guided me during study.

I show my sincere gratitude to my Parents Sri D.V. Hadimani, Smt

Shashikala my sisters Miss Shilpa, Miss Vijaya for their kind moral support

throughout my carrier.

I also thankful to the librarians Mrs.G.C.Gull & Miss. Vyshali for their

needy help and also thankful to Mr. Mallesh, Mr. Mahantesh, and Mr. Vinayak

for their kind cooperation during this work.

Last but not the least I take this opportunity to thank all those non teaching

staff, KLE’s Ayurveda pharmacy staff, Maratha Mandal Microbiology dept staff,

KLE’S Engineering college staff, Friends of KLE’S Pharmacy College, Librarian

for their unforgettable help through out the course of study.

Date:

Place: Belgaum Dr RAGHUEER B.A.M.S

ABBREVIATIONS

% - Percentage

+ve - Positive

-ve - Negative

e.g. - Example

Hrs - Hours

Mcg - Microgram

mg - Milligrams

mL - Micro litre

mm - Millimeter

Na - Sodium

Mg - Magnesium

K - Potassium

Fe - Ferrum

R.T. - Rasa Tarangini

R.R.S - Rasaratna Samuchaya

w/w - Weight by weight

Wt. - Weight

i.e - That is

Navasadara satva (I) – Navasadara satvapatana by using market sample

Navasadara satva (II) – Navasadara satvapatana by using chullika lavan

MIC - Minimal inhibitory concentration

MBC - Minimum bactericidal concentration.

E.Coli - Escherichia Coli

S. aureus - Staphylococcus Aureus

K.pneumoniae - Klebsiella pneumoniae

S.pneumoniae - Staphylococcus pneumoniae

ABSTRACT

Navasadara is one of the Sadharana rasa mentioned in Rasashastra texts. Satva

of Navasadara is mentioned only in the text Rasatarangni and it is indicated in

Amlapitta, Hrudaya dourbalya, Ruchivardhaka, Murcha, Puppusashotha,

Navasadara (Ammonium Chloride; NH4Cl) is considered under Lavana

varga by some authors and some have included it under kshara varga. Chullika lavana

has been mentioned as the one of the source of Navasadara and in some context

chullika lavana has been used synonymous with Navasadara. Extraction procedure of

Navasadra from Chullika lavana mentioned in text Rasa Ratna samucchaya but no

effort made till date to extract the Navasadara from chullika lavana because of easy

availability of Navasadara in market. Navasadara samples available in the market are

synthetically prepared hence the desired clinical effects are not obtained hence testing

the genuinity of Market samples with self prepared Navasadara on Analytical

parameters is also a need of today.

Being Kshara Navasadara is said be to be having the Antimicrobial property

so both samples were subjected for antimicrobial studies using Disc diffusion and

MIC methods. Hence this study is conducted to screen the Genuinity of market

samples of Navasadara and comparing with self prepared sample on Analytical and

Antimicrobial property parameters.

RESULTS: Navasadara satva not obtained from the Chullika lavana, but market

sample yield good amount of Satva after subjecting for Satvapatana as per the

reference Rasatarangini. Antimicrobial study revealed both samples has shown

sensitive against all organisms except E coli. But market sample has shown

bactericidal effect against E coli and Staph pneumoniae.

Keywords: Navasadara, Chullika lavana, Satvapatana, Antimicrobial study.

TABLE OF CONTENTS

SI. NO.

TITLE

PAGE

NO 1 INTRODUCTION 1-2 2 AIMS & OBJECTIVES 3 3 REVIEW OF LITERATURE 4-32 3.1 NAVASADARA 4-17 3.2 KHATIKA 18-23 3.3 SATVAPATANA 24 3.4 REVIEW OF MICROBIOLOGY 24-32 4 METHODOLOGY 33-58 4.1 CHULLIKA LAVANA SHODHANA 34-35 4.2 NAVASADARA SHODHANA 35-36 4.3 KHATIKA SHODHANA 36-37 4.4 NAVASADARA SATVAPATANA (I) 38-39 4.5 NAVASADARA SATVAPATANA (II) 39-40 4.6 DETERMINATION OF pH 41 4.7 DETERMINATION OF LOD 41 4.8 DETERMINATION OF ASH VALUE 42 4.9 DETERMINATION OF ACID INSOLUBLE ASH 43 4.10 DETERMINATION OF SPECIFIC GRAVITY 44 4.11 DETERMINATION OF SOLUBILITY 44 4.12 QUALITATIVE ANALYSIS OF SAMPLES 48 4.13 ANTIMICROBIAL METHODS 48 5 RESULTS 59-66 5.1 OBSERVATIONAL RESULTS 59-60 5.2 ANALYTICAL RESULTS 60-63 5.3 ANTIMICROBIAL RESULTS 63-66 6 DISCUSSION 67-69 7 CONCLUSION 70 8 SUMMARY 71-72 9 BIBILOGRAPHY 73-76 10 ANNEXURE

LIST OF TABLES

TABLE.NO

NAME OF TABLE

PAGE

NO

1 Difference of opinions about Navasadara 4

2 Synonyms of Navasadara 6

3 Ammonium Chloride (NH4Cl) –Physical & chemical

properties

10

4 Synonyms of Khatika 19

5 Khatika- physical and chemical properties 21

6 Organoleptic characters of raw materials 59

7 Chullika lavana shodhana 59

8 Navasadara shodhana 59

9 Khatika shodhana 60

10 Navasadara satvapatana (I) 60

11 Navasadara satvapatana (II) 60

12 Raw material analysis 60

13 Physico-chemical parameters 61

14 Solubility 61

15 Qualitative Analysis 62

16 Quantitative Analysis 62

17 AES-ICP Result 63

18 Results of cup-plate /disc diffusion method 63

19 Results of MIC 65

20 Result of MBC method 66

LIST OF PHOTOGRAPHS Plate No

Name of Plates

1 Brick kiln, Chullika lavana, Navasadara, Khatika

2 Navasadara shodhana

3 Chullika lavana shodhana

4 Khatika shodhana

5 Navasadara satvapatana (I)

6 Navasadara satvapatana (II)

7 Antimicrobial equipments

8 Antimicrobial study - MIC Method

9 Antimicrobial study - Disc diffusion Method



12 Antimicrobial study – MBC Method

LIST OF FIGURES

Sl.No. Figure

Page No

01 Chemical Structure of Ammonium Chloride

12

02 Lab preparation of ammonia

16

ANNEXURE

Sl.No. Annexure

01 Authentification letter

02 Analytical report

03 XRD-Analysis

04 ICP-AES report

Introduction

INTRODUCTION

Rasashastra is a new development in Ayurveda, as it is not mentioned in

traditional eight specialties i.e. Ashtanga Ayurveda. Rasashastra is a branch of

Ayurveda which deals with the various pharmaceutical processes such as shodhana,

marana, jarana, murchana, satwapatana etc. and description of metals, minerals,

animal products & poisonous herbal drugs, most precious things like gold, gems etc

& their therapeutic usage. These drugs are categorized or classified in different

groups like Maharasa, Uparasa, Sadharanarasa, Dhatu, Upadhatu, Ratna and so on.

Navasadara is one of the rasadravya included in Sadharana rasa1 group

of drug. Explanation of Navasadara is found in all most all Rasagranthas.

Rasendramangala the earliest book to mention about Navasadara and used as

dhatuvadartha, in parada jarana. But the amayika prayoga of Navasadara is explained

in the text Rasahridaya tantra in 8th century. Process of Navasadara satvapatana is

only explained in the text Rasatarangini2. Navasadara is used as a one of the jarana

drvya, i.e used in parada jarana and also used as lohadraavanaartha.

There is difference of opinions regarding Navasadara. Some of them explained

that it is one of the kshara and some are explained that it is a type of lavana3. To rule

out these differences of opinions proper identification by classical and modern

method, pharmaceutical, pharmacological study should be carried out.

While explaining the occurance of Navasadara it is told that the Chullika

lavana or ishtika lavana is the source for Navasadara i.e we can extract Navasadara

from this chullika lavana or ishtika lavana4.

“A Comparative Antimicrobial study of Navasadara Satva, by taking two different samples

of Navasadara”

1

Introduction

It is also extracted from Naramootra, Oustra mutra, oustra mala etc. In Punjab

it is extracted from mud5. As Navasadara is indicated as netrya, shweta kustahara,

deepana, kaphanissaraka, puppusashothahara,etc6. To assess its efficacy, its

antimicrobial study is carried out on gram positive and gram negative organisms.

. This study has been undertaken in three headings. A) Preparation of

Navasadara satva as per Rasatarangini, B) Comparative physico chemical analysis of

both raw samples as well as final product, and C) Comparative antimicrobial activity

of Navasadara satva using Disc Diffusion and MIC methods .

All the results obtained out of Analytical and Antimicrobial studies has been

tabulated and compared among them as per set of parameters.

Chullika lavana doesn’t yield Satva but the market sample given good amount

of Satva after subjecting both samples for satvapatana as per the reference

Rasatarangini. Antimicrobial studies revealed that both samples shown sensitive

against all four organisms except E-coli indicating good bacteriostatic action. Market

sample proven to be having good bactericidal action against E-coli and Staph

pneumoniae.

“A Comparative Antimicrobial study of Navasadara Satva, by taking two different samples

of Navasadara”

2

Aims and Objectives

AIMS AND OBJECTIVES

1. Preparation of Navasadara Satva by using both Chullika lavana and Navasadara

2. To screen Antimicrobial activity of Navasadara Satva.

3. Procurement of Chullika lavana from brick kiln.

4. Collection of Navasadara and Khatika.

5. Identification of Navasadara and Khatika according to grahya laxanas.

6. Shodhana of Chullika lavana, Navasadara, Khatika.

7. Physicochemical analysis of-

(a) Raw materials- Chullika lavana, Navasadara, Khatika

(b) Shodhita - Chullika lavana, Navasadara, Khatika

(c) Navasadara Satva

“A comparative Antimicrobial study of Navasadara Satva by taking two different samples Of Navasadara”

3

Review of Literature

REVIEW OF LITERATURE

3.1 NAVASADARA

Historical Review:

In vedakala and samhita kala there is no explanation about Navasadara. But

first it was mentioned in the text Rasendramangala in 7th century7. But it was used

only for lohavedartha. Later on during 8th century it was used for dehaveda by

Rasahrudaya tantrakara. Later on all rasa granthas have explained about this drug.

In Rasa Granthas:

• Among Rasa granthas, a reference regarding Navasadara is found in

Rasahridaya tantra8, grouped under lavana varga.

• Rasakamadhenu gives more detailed explanation regarding special varieties.

• Difference of openions were found regarding Navasadara, some authers

included under uparasa, some are included under sadharana rasa, lavana varga,

kshara varga, shown below.

Table No.1: Difference of openions about Navasadara

Sl.No. Reference Uparasa Sadharana

Rasa

Lavana

Varga

Kshara

Varga

01 Rasahridaya tantra - - + -

02 Rasarnava9 - - + -

03 Anandakanda10 + - + +

04 Rasendrachudamani - + + +

05 Rasendrasara

Samgraha11

- - + -

06 Rasaratna Samuchaya - + + +

“A comparative Antimicrobial study of Navasadara Satva by taking two different samples of Navasadara”

4


07 Ayurveda Prakash12 - + + +

08 Rasendra Purana13 - + + +

09 Rasajala Nidhi14 + - - -

10 B.Ra.Ra.Sundara15 - - + +

11 Rasamrita16 - - + -

12 Nighantu Ratnakara17 - - + +

Difference of opinion found regarding Navasadara. In ancient period acharyas

use to prepare Navasadara in many ways. According to rasaratna samuchaya, it is

explained that after burning Karira (Capparis decidua Edgew) and Pilu (Salvadora

persica Linn) kasta, by using that bhasma we can extract Navasadara. There is an

explaination regarding istika lavana or chullika lavana in many Rasa texts like

Rasendra chudamani, Rasajalanidhi, Ayurveda prakash, Rasendra purana, brihat

rasaraja sundara etc. The white part remained after burning in brik kiln is said to be as

a chullika lavana or istika lavana, it is considered as Navasadara. In Punjab it is

prepared by using mud, in Egypt it is prepared by using oustra mala (Camel excreta),

in Europe it is prepared by using naramutra (urine of human being), so it is named as

Narasara or Navasadara. In Itally, Sisilly it is found near volcano as a native form.

VERNACULAR NAMES18

1) Samskrit - Navasara, Narasara, Nrisara, Chullika lavana, Kshara sreeshta

2) English - Ammonium Chloride (Nh4Cl)

3) Hindi -Nousaadar

4) Kannada - Navasaagara

5) Gujarat - Navasaara

6) Telugu - Navasaaram


5


7) Tamil - Navachchaaram

8) Konkani - Navasagar

9) Punjabi - Nousadar

10) Pharsi - Noushadar

11) Malayalam - Navasaagaram

12) Latin - Ammonium chloride

13) Marathi -Navsaagar

14) Bengali -Nishaadal

Table No.2: Synonyms of Navasadara19

Sl. No. Synonyms Sl. No. Synonyms

1. Amritakshara 16 Navasagah

2. Kittakshara 17 Navasagarah

3. Ksharasreshta 18 Navasadaram

4. Gojam 19 Navasarah

5. Gomalam 20 Navyasarah

6. Chooli 21 Nishaadala

7. Choolikah 22 Nousadara

8. Choolikaah 23 Nrirasara

9. Chullika lavana 24 Nrisadaka

10. Chulika lavana 25 Nrisadarah

11. Chulhika 26 Nrisarah


6


12. Narasara 27 Vajrakah

13. Navasadara 28 Vajrakshara

14. Bida 29 Vajrakakshara

15 Bida lavana 30 Vidaarakam

ORIGIN

In ancient period it was prepared by using naramutra, exreta of animals, ash

obtained after burning Karira and pilu kasta (it is called as chullika lavana). Found

near volcano in native form. At present it is prepared artificially in laboratory.

OCCURANCE

Navasadara is available in two major forms as swabhavika and kritrima.

Swabhavika is chullika lavana i.e. which is available naturally by burning wood.

Kritrima is artificislly prepared in laboratory.

UTPATTI STHANA: Egypt, Europe, Madhya Asia, Italy, Sisily, Punjab, Haryana,

Rajasthan, and Gujarat.

TYPES:

Types of Navasadara are first mentioned in the text Rasakamadhenu (16th

century), in that, author mentioned two types of Navasadara, but detail description is

not available.

(i) Yogambari: It is said to be uttama (good variety).

(ii) Chullika

Two types according to the text Ayurveda Prakash

(i) Kritrima (Artificial)


7


(ii) Akritrima (Natural)

Navasadara which is obtained by the mutra, purisha of manushya, pashu is called

as akritrima.

On the base of Praaptisthana – Two types

(i) Khanija

(ii) Vaanaspatika

GUNA: (RRS 79)

Paandura

Lavana

Laghu

R.R.S.3/129 - Deepana, Pachana, Laghu, Sara, Teekshna, Sukshma, Bhukta

mamsajarana, Parada jarana (sarvottama), lohadravanartha.

PHARMACOLOGICAL PROPERTIES AS PER RASATARANGINI:

Rasa – Lavana

Guna – Snigdha, sukshma, laghu, teekshna

Veerya – Ushna

Doshagnata – Tridoshaghna

Karma – Saaraka, pachaka, agnideepaka, mamsaajirnanivaaraka

Rogaghnata – Puppusashotha, Murcha, Aruchi, Shoola, Udararoga, Malastamba,

Pleeha, vrina vidaraka.

Gulma, Mukhashosha, Adhmana, Kaphanashaka (R.R.S.3/129)

• mÉëÌiÉvrÉÉrÉãwÉÑ ÍvÉUxÉ: mÉÏQûlÉã lÉuÉxÉÉSUqÉç -

uÉæ±cÉÎlSìMüÉ


8


Navasadara is indicated for Pratisyaya and shira shoola by Vaidya chandrika

Method of extraction:

• SÕÌiÉ uÉæ± cÉÎlSìMüÉã£ãü cÉÔhÉïiÉÉãrÉÉprÉÉÇ

RûÉãsÉÉrÉl§ÉãhÉ mÉÉÍcÉiÉã YuÉÉjÉqÉãSã lÉuÉxÉÉSUqÉmrÉ§É |

-Vachaspatyam vol 5

Explanation regarding Navasadara in Vachaspatyam is, Navasadar churna is

dissolved in water and pachan is carried out in Dolayantra to get shuddha Navasadara.

NAVASADARA SHODHANA:

According to various Rasagranthas written by different rasavaidyas,

navasadara shodhana is mentioned in various methods.

lÉuÉxÉÉUliÉÑ xÉÍsÉsÉå Ì§ÉaÉÑhÉå SìÉuÉrÉåÎ°wÉMçü |

uÉx§ÉmÉÔiÉqÉç iÉiÉ: M×üirÉÉ pÉÉeÉlÉå xjÉÉmÉrÉå¨ÉiÉ: ||

cÉÑÎssÉMüÉrÉÉqÉç ÌlÉkÉÉrÉÉjÉ mÉcÉå¨ÉÏuÉëÉÎalÉlÉÉ

pÉ×zÉqÉ |

eÉsÉÇ zÉÑwMÇü iÉÑsÉxrÉ¶É lÉ×xÉÉUÇ ÌuÉqÉsÉÇ WûUåiÉç ||

- RT14/3-4

1 part of Navasadara is dissolved completely in 3 parts of jala, and then it is

filtered. This filtrate is kept on intense heat. Heat is given until all water part get

evaporates. Then it is collected.

lÉuÉxÉÉUÉã pÉuÉãcNÒûkSÇ ¶ÉÔhÉïiÉÉãrÉæÌuÉïmÉÉÍcÉiÉ: |

RûÉãsÉÉrÉl§ÉãhÉ rÉ¦ÉãlÉ ÍpÉwÉÎapÉrÉÉãïaÉÍxÉkSrÉã || -

Rasendra sambhava


9

Navasadara is made into churna mixed with jala and pachana is carried out in

Dolayantra to purify.


According to Parada samhita –

Navasadara is taken in khalwa yantra, and bhavana is given with Jambeera

swarasa / Nimbu swarasa, then it is dried in sun light, after complete drying it is kept

in urdwa paatana yantra. Then collect the shuddha Navasadara from inner part of the

upper pot.

According to Rasaratna Samuchaya – Navasadara is dissolved in boiling

water, and then filtered through cloth, dried. Thus formed navasadara is shodhita.

NAVASADARA YOGAS:20

1) Gulmari rasa 10) Navasara churna yoga

2) Kshara parpati 11) Talakeshwara rasa,

3) Vrishchikdamshahara lepa. 12) Kshaya kesari rasa,

4) Pleehari vati 13) Amlapittantaka rasa

5) Talasindhura 14) Navasadara druti

6) Meghanada rasa 15) Lavanadya churna,

7) Netrasudha anjana 16) Tripura bhairava rasa

8) Jwarashara arka 17) Deepana churna

Table No. 3: Ammonium Chloride (NH4Cl) –Physical & chemical properties21

IUPAC Name Ammonium Chloride

Other Name Sal ammoniac

Molecular formula NH4Cl

Molar mass 53.49g/mol

Appearance White solid

Density 1.5274g/cm3

Melting Point 3380 C


10


Solubility in water 29.7 g/100ml (00C)

37.2 g/100ml (200C)

77.3 g/100ml (1000C)

Solubility in alcohol 0.6 g/100ml (190C)

Acidity 9.245

Refractive index 1.642

Standard enthalpy of formation -314.55 kJ/mol

Standard molar entropy 94.85 JK-1 /mol

Flash point Non flammable

LD50 1650 mg/kg, oral (rat)

Anions Ammonium fluoride Ammonium bromide Ammonium iodide

Cations Sodium chloride Potassium chloride Hydroxylamonium chloride

Ammonium chloride (NH4Cl) (also Sal Ammoniac, salmiac, nushadir

salt, sal armagnac, sal armoniac, salt armoniack) is, in its pure form, a clear white

water-soluble crystalline salt of ammonia. The aqueous ammonium chloride solution

is mildly acidic. Sal ammoniac is a name of natural, mineralogical form of ammonium

chloride. The mineral is especially common on burning coal dumps (formed by

condensation of coal-derived gases), but also on some volcanoes.

CHEMICAL STRUCTURE OF AMMONIUM CHLORIDE:


11

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Chlorine

http://en.wikipedia.org/wiki/Salt

http://en.wikipedia.org/wiki/Ammonia

http://en.wikipedia.org/wiki/Sal_ammoniac

http://en.wikipedia.org/wiki/Coal


Fig.No 1. Chemical structure of ammonium chloride

HISTORY:

The Romans called the ammonium chloride deposits they collected from near

the Temple of Jupiter Amun (Greek �μμων Ammon) in ancient Libya 'sal

ammoniacus' (salt of Amun) because of proximity to the nearby temple. Salts of

ammonia have been known from very early times; thus the term Hammoniacus sal

appears in the writings of Pliny, although it is not known whether the term is identical

with the more modern sal-ammoniac.

In the form of sal-ammoniac, ammonia was known to the alchemists as

early as the 13th century, being mentioned by Albertus Magnus. It was also used by

dyers in the middle Ages in the form of fermented urine to alter the colour of

vegetable dyes. In the 15th century, Basilius Valentinus showed that ammonia could

be obtained by the action of alkalis on sal-ammoniac. The Haber process to produce

ammonia from the nitrogen in the air was developed by Fritz Haber and Carl Bosch in

1909 and patented in 1910. It was first used on an industrial scale by the Germans

during World War I.


12

http://en.wikipedia.org/wiki/File:NH4Cl.png


Sal Ammoniac was named after it was observed in the Temple of Zeus-

Ammon in Egypt; its name means "salt of Ammon". It was the white crystalline

substance that remained on the ceiling and walls after camel dung was burned. The

modern name "ammonium" comes from Sal Ammoniac. There are a few stories of

Alexander the Great finding such crystals in the coal seams of Tajikistan. The

substance was known as nao sha in China, nao sadar in India, and nushadir in Persia

and Arabic countries.

Sources:

The substance occurs naturally in volcanic regions, forming on volcanic

rocks near fume-releasing vents. The crystals deposit directly from the gaseous state, and tend

to be short-lived, as they dissolve easily in water. It is a by-product of the Solvay process used

to produce sodium carbonate.

Ammonium chloride is prepared commercially by reacting ammonia (NH3) with hydrogen

chloride (HCl). As these chemicals are corrosive, this process has to be performed in vessels

lined with nonreactive materials (e.g. glass, enamel,lead, or PVC).

NH3 + HCl → NH4Cl

SYNTHESIS AND PRODUCTION:

Because of its many uses, ammonia is one of the most highly produced

inorganic chemicals. About 80% or more of the ammonia produced is used for

fertilizing agricultural crops.

Before the start of World War I, most ammonia was obtained by the dry

distillation of nitrogenous vegetable and animal waste products, including camel

dung, where it was distilled by the reduction of nitrous acid and nitrites with


13

http://en.wikipedia.org/wiki/Volcano

http://en.wikipedia.org/wiki/Solvay_process

http://en.wikipedia.org/wiki/Sodium_carbonate

http://en.wikipedia.org/wiki/Ammonia

http://en.wikipedia.org/wiki/Hydrogen_chloride

http://en.wikipedia.org/wiki/Hydrogen_chloride

http://en.wikipedia.org/wiki/Glass

http://en.wikipedia.org/wiki/Vitreous_enamel

http://en.wikipedia.org/wiki/Lead

http://en.wikipedia.org/wiki/PVC

http://en.wikipedia.org/wiki/World_War_I

http://en.wikipedia.org/wiki/Dry_distillation

http://en.wikipedia.org/wiki/Dry_distillation

http://en.wikipedia.org/wiki/Camel

http://en.wikipedia.org/wiki/Manure

http://en.wikipedia.org/wiki/Distillation

http://en.wikipedia.org/wiki/Nitrous_acid

http://en.wikipedia.org/wiki/Nitrite


hydrogen; in addition, it was produced by the distillation of coal, and also by the

decomposition of ammonium salts by alkaline hydroxides such as quicklime, the salt

most generally used being the chloride (sal-ammoniac) thus:

2 NH4Cl + 2 CaO → CaCl2 + Ca (OH) 2 + 2 NH3

Medical Properties and Physiological Action:

Ammonia gas is very alkaline, and an irritant to mucous surfaces. Inhaled, it

causes an overpowering sense of suffocation and spasm of the glottis, and when

prolonged, violent inflammation of the air-passages. Solution of ammonia when

swallowed causes destructive inflammation of the mucous membrane, extending to

the stomach. The long-continued use of ammonia interferes with digestion by

neutralizing the gastric juice, and by increased waste of tissue causes pallor,

Emaciation and feebleness. In the blood it injures the red blood globules, and thus

affects the nutrition of the body, being largely converted into urea. The preparations

of ammonia are stimulant expectorants.

Aqua ammonia is administered by inhalation in syncope and shock, and as a

counter-irritant; for which purpose ammonia liniment is also employed. The

incautious inhalation of ammonia may cause inflammation of the fauces and glottis,

but when cautiously employed sometimes gives relief to acute catarrh and hay

asthma. The diluted aqua ammonia will relieve the pain of stings of insects, and the

strong aqua ammonia is an antidote, when at once applied, to the bite of venomous

snakes, and of rabid animals. The aromatic spirits of ammonia is useful in acidity of

stomach, gaseous eructations and abdominal distensions; also in sick headache and

migraine; but the bromides are more effective in the latter affection. Ammonia salts


14

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Coal

http://en.wikipedia.org/wiki/Alkaline

http://en.wikipedia.org/wiki/Calcium_oxide

http://en.wikipedia.org/wiki/Ammonium_chloride


stimulate the liver and increase the secretions of the kidneys and intestinal mucous

glands and the action of the heart, hence are frequently used in adynamic states,

constipation, coated tongue and scanty urine.

Ammonia and Its Salts (NH3):

When pure, ammonia is a colourless gas, capable of being liquified; of very

pungent odour, the fumes producing an alkaline reaction; it forms salts with acids,

but always takes an atom of basic water, and hence by most chemists these salts are

regarded as containing an oxide of a hypothetical metal called ammonium (Nh4);

thus sal ammoniac may be regarded as a hydrochlorate of ammonia (Nh3, Hc1) or

chloride of ammonium (Nh4 Cl). Ammonia also forms direct combinations with

acids, as carbonic acid, not true salts; a compound of carbonic acid and ammonia

(Nh3, Co2) is perhaps present in the sesquicarbonate. Gaseous ammonia is

sometimes made use of therapeutically, evolved usually when thus employed, from

liquor ammonias, in which it is contained.

Laboratory Preparation of Ammonia

For use in the laboratory, ammonia is prepared by heating an ammonium salt

with a strong base. It can also be prepared by reacting a metal nitride with water. All

ammonium salts heated with alkali evolve ammonia. Ammonium nitrate is not used in

laboratory preparation, since it is explosive in nature and may decompose forming

nitrous oxide and water vapour.


15


Fig No.2 Ammonia is prepared in the laboratory by heating a mixture of solid

ammonium chloride and calcium hydroxide.

Reactants: Ammonium chloride and calcium hydroxide, Ratio 2:3

Procedure: Ammonium chloride and calcium hydroxide are ground together and

heated slowly in a round bottomed flask with its neck sloping downwards. The gas

is passed through a drying tower before collection.

Collection: Ammonia gas is lighter than air and hence collected by the downward

displacement of air. It is not collected over water since it is highly soluble in water.


16


DETECTION OF AMMONIA:

Ammonia and ammonium salts can be readily detected, in very minute traces, by the

addition of Nessler's solution, which gives a distinct yellow coloration in the

presence of the least trace of ammonia or ammonium salts.

Nessler's reagent: In analytical chemistry Nessler's reagent is a reagent used to detect

small concentrations of ammonia. A yellow coloration indicates the presence of

ammonia. At high concentrations, a brown precipitate may form.

Larger quantities can be detected by warming the salts with a caustic alkali or

with quicklime, when the characteristic smell of ammonia will be at once apparent.

The amount of ammonia in ammonium salts can be estimated quantitatively

by distillation of the salts with sodium or potassium hydroxide, the ammonia evolved

being absorbed in a known volume of standard sulfuric acid and the excess of acid

then determined volumetrically; or the ammonia may be absorbed in hydrochloric

acid and the ammonium chloride so formed precipitated as ammonium chlorplatinate,

(NH4)2PtCl6.

Test: Mixture + NaOH Boil and hold a wet litmus paper at the mouth of

the test tube.

Observation: Gas with ammonia smell having alkaline action on litmus paper

and white dense fumes with glass rod dipped in con HCl NH4 present

Toxicity:

It is toxic if swallowed, inhaled or absorbed through the skin. It presents

neurological hazard and may act as a carcinogen and be a reproductive hazard. It is


17


corrosive and causes burns. Always wear safety glasses and gloves and manipulate in

a well ventilated environment.

3.2 KHATIKA

Historical review –

In Vedas

There is no description about Khatika in Vedas.

In Samhitas

A little description is available in Samhitas.

In Rasagranthas

• Bhava Misra has included Khatika in Uparasa Varga.

• Yasodhara Bhatta in Rasa Prakasa sudhakara has mentioned the Khatika for

the preparation of Rasa karpura. He has also used khatika for the shodhana of

Swrna. He has used the word khatika to describe the white variety of sulphur.

• Vagbhatta in his text book Rasaratna samucchaya has also used the word

Khatika to describe the white variety of sulphur.

• In Ayurveda Prakash, Acharya Madhava.has included khatika in Uparasa

group. He also described the synonyms, varieties and therapeutic indications

of Khatika.

• Sri Sadananda Sharma in his text Rasa Tarangini has described the synonyms,

varieties and therapeutic indications of Khatika along with its purification

method.


18


Table No. 4: Synonyms:22

Sl. No. Synonyms 1. Khatika

2. Khatini.

3. Khati

4. Lekhana mrittika

5. Khatini

6. Khatinika

7. Varnika

8. Varnalekhika

VERNACULAR NAMES: 23

Sanskrit - Khatika

Hindi - Khariya

Kannada - Sunna

English - Chalk, Pipe clay

Marathi - Khadu

Telugu - Sime sunnam.

Gujarathi - Khadi, Khadimati.

Bengli - Phula khadi.

OCCURRENCE: 24

Chalk is found in the form of marble, limestone, calcite or island spar in

nature. Pipe clay is one of these varieties. The chalk is a soft, fine grained limestone.

It was formed as a mud on the bottom of an ancient sea. It differs from pure, fine

grained lime stones only in still being soft and easily rubbed off. That is, it did not

change in to hard rock. The white cliffs of Dover are thick layers of Chalk.


19


TYPES:25

Khati - Dusty white in colour.

Goura khati - Mridu pashana sadrusa white in colour.

Grahya lakshana26 of Khatika is soft, White or dull dusty white color, and heavy in

weight like stone, brittle and stics to tongue.

PHARMACOLOGICAL PROPERTIES:27

Rasa – Madura, Tikta.

Guna - Sheeta

Virya - Sheeta

Doshagnata - kapha, Pittahara.

Karma - Grahi, Dahahara.

THERAPEUTIC USES:28

Kasa, Haridravarnatisara, Atisveda, Netra roga, Sotha, Vruna, Pravahika,

Grahani, Raktapitta.

Dose - 1 Masha.

Anupana - Sheetalajala.

Formulations-

Khatikadi peya, Dashana sanskar churna, Mugdharasa.

Shodhana method29

For Shodhana it should be mixed with water and filtered through cloth, and

allowed to remain undisturbed for 3-4 hours. Decant the water and collect the material

settled at the bottom. Decant the water and collect the material settled at the bottom.

KHATIKA SHODHANA.

Reference:


20


ZÉOûÏcÉÔhÉïÇ vÉÑ®mÉÉ§Éã ÌlÉkÉÉrÉ ÌuÉqÉsÉã eÉsÉã |

mÉë¤ÉÉsÉrÉãÌ²kÉÉlÉalÉÉã ÌuÉvÉÑkrÉÌiÉ lÉ xÉÇvÉrÉ: || - R

T 11/210

Khatika is purified by nirmalikarna process by using water. 1 part of khatika

churna is dissolved in 4 parts of water kept for some time and filtered through cloth

and dried in sunlight and collected.

Table No.5: Khatika physical, chemical properties30:

Name Calcium carbonate

Chemical formula CaCO3

Appearance White solid

Formula weight 100.1 amu

Melting point Liquifies under high pressure at1612K (13390C)

Boiling point Decomposes at 1172 K (8990C)

Density 2.7 × 103 kg/m3

Crystal structure Calcite, aragonite, or vaterite

Solubility 0.0013gm in 100gm water

Standard enthalpy formation liquid -1154 kJ/mol

Standard enthalpy formation solid -1207 kJ/mol

Standard molar enthalpy 93 J/(mol K)

CHALK:

Chalk is a soft, white, porous form of limestone composed of the mineral

calcium carbonate. It is relatively resistant to erosion and slumping compared to the

clays that it is usually associated with, and so forms tall steep cliffs where chalk


21

http://www.chemistrydaily.com/chemistry/Limestone

http://www.chemistrydaily.com/chemistry/Mineral

http://www.chemistrydaily.com/chemistry/Calcium_carbonate

http://www.chemistrydaily.com/chemistry/Erosion

http://www.chemistrydaily.com/chemistry/Clay

http://www.chemistrydaily.com/chemistry/Cliff


ridges meet the sea. Chalk hills, known as chalk downland, usually form where bands

of chalk reach the surface at an angle. Because chalk is porous, chalk downland

usually holds a large water table, providing a natural reservoir that releases water

slowly through dry seasons.

Chalk has been quarried from prehistory, providing building material and marl

for fields. In southeast England, deneholes are a notable example of ancient chalk pits.

Blackboard chalk is a substance used for drawing on rough surfaces, as it

readily crumbles leaving particles that stick loosely to these surfaces. Blackboard

chalk, often supplied in sticks about 5 cm long, is not actually made from the mineral

chalk but from gypsum (calcium sulfate). Similarly, the "chalk" used by tailors is

usually made from talc (magnesium silicate).

CHALK FORMATION:31

The Chalk Formations of Europe are thick deposits of chalk, a soft porous

white limestone, deposited in a marine environment during the upper Cretaceous

period. They appear most prominently in England. The formations are divided into

three parts: The Upper Chalk, the Middle Chalk, and the Lower Chalk. The famous

White cliffs of Dover, England are a good example of a Chalk Formation deposit.

Another good example displaying the sequence of the Chalk Formation are the

southern cliffs on the Isle of Wight, England and the quarries and motorway cutting at

Blue Bell Hill, Kent, England (which has been classified as a Site of Special Scientific

Interest).


22

http://www.chemistrydaily.com/chemistry/Hill

http://www.chemistrydaily.com/chemistry/Downland

http://www.chemistrydaily.com/chemistry/Water_table

http://www.chemistrydaily.com/chemistry/Marl

http://www.chemistrydaily.com/chemistry/England

http://www.chemistrydaily.com/chemistry/Denehole

http://www.chemistrydaily.com/chemistry/Blackboard

http://www.chemistrydaily.com/chemistry/Cm

http://www.chemistrydaily.com/chemistry/Gypsum

http://www.chemistrydaily.com/chemistry/Calcium_sulfate

http://www.chemistrydaily.com/chemistry/Talc

http://www.chemistrydaily.com/chemistry/Magnesium

http://www.chemistrydaily.com/chemistry/Silicate

http://www.chemistrydaily.com/chemistry/Europe

http://www.chemistrydaily.com/chemistry/Chalk

http://www.chemistrydaily.com/chemistry/Limestone

http://www.chemistrydaily.com/chemistry/Cretaceous


http://www.chemistrydaily.com/chemistry/White_cliffs_of_Dover


http://www.chemistrydaily.com/chemistry/Isle_of_Wight


http://www.chemistrydaily.com/chemistry/Quarry

http://www.chemistrydaily.com/chemistry/Kent



• Chemistry: CaCO3, Calcium Carbonate

• Class: Carbonates

• Group: Calcite

• Uses: In cements and mortars, production of lime, limestone is used in the

steel industry; glass industry, ornamental stone, chemical and optical uses and

as mineral specimens.

Calcite, which gets its name from "chalix" the Greek word for lime, is a

most amazing and yet, most common mineral. It is one of the most common minerals

on the face of the Earth, comprising about 4% by weight of the Earth's crust and is

formed in many different geological environments. Calcite can form rocks of

considerable mass and constitutes a significant part of all three major rock

classification types. It forms oolitic, fossiliferous and massive limestones in

sedimentary environments and even serves as the cements for many sandstones and

shales. Limestone becomes marble from the heat and pressure of metamorphic events.

Calcite is even a major component in the igneous rock called carbonatite and forms

the major portion of many hydrothermal veins. Some of these rock types are

composed of better than 99% calcite.

The crystals of calcite can form literally a thousand different shapes by

combining the basic forms of the positive rhombohedron, negative rhombohedron,

steeply, moderately and slightly inclined rhombohedrons, various scalahedrons, prism

and pinacoid to name a few of the more common forms. There are more than 300

crystal forms identified in calcite and these forms can combine to produce the

thousand different crystal variations. Calcite also produces many twin varieties that

are favorites among twin collectors. There are also phantoms, included crystals, color


23

http://mineral.galleries.com/rocks/limestone.htm

http://mineral.galleries.com/rocks/sedimentary.htm

http://mineral.galleries.com/rocks/sandstone.htm

http://mineral.galleries.com/rocks/shale.htm

http://mineral.galleries.com/rocks/marble.htm

http://mineral.galleries.com/rocks/metamorphic.htm

http://mineral.galleries.com/rocks/igneous.htm

http://mineral.galleries.com/rocks/carbonatite.htm


varieties, pseudomorphs and unique associations. There simply is no end to the

varieties of calcite.

3.3 SATVAPATANA:32

The term satvapatana is composed of two words, satva and patana. Satva is an

essence of drug, wher as patana means to extract.

Shloka:

¤ÉÉUÉqsÉ mÉëÉuÉMæürÉÑï£üÇ kqÉÉiÉÉqÉÉÇ MüU

MüÉã¹Mãü |

rÉ¦ÉiÉÉã ÌlÉaÉïiÉ: xÉÉU: xÉiuÉÍqÉirÉÍpÉSÏrÉiÉã || - Rasadarpana

The drugs of mineral origin when mixed with alkali (kshara), acid (amla) and

fusing materials (dravaka gana) chakrikas prepared and subjected to intense heat in a

furnace, it will liberates the metallic portion of the material is called satva.

History of Satvapatana:

In Vedic literature metals like gold, silver, iron etc are mentioned. Mention of

these metals which are not available in Free State in nature means people of Vedic era

were having knowledge of satvapatana. Acharya Nagarjuna in 7th century AD

described the process in details in his book Rasendramangala. The research workers

of Rasashastra have developed newer techniques decided the fuel and various signs

which indicated the melting of mineral substances and separation of metallic contents.

Also various types of fuels, equipments and furnces are mentioned in these ancient

texts. Further the literature of Rasashastra describes the form appearance, colour, and

etc. characters of satva bhasmeekarana process and uses of satva.

3.4 REVIEW OF MICROBIOLOGY: 33

Medical microbiology is the study of microbes that infect humans, the

diseases, which they cause, their diagnosis, prevention and treatment. As microbes are


24


invisible to the unaided eye, definitive knowledge about them had to await the

development of microscopes.

Microbiology a branch of science, dealing with microorganisms. These

microorganisms could be pathogenic or nonpathogenic with varied applications.

Some of them play an important role in maintaining the ecological balance

on earth. Some of them have commercial applications as in synthesis of chemical

products and food, some others are pathogenic that is disease causing.

The group of microbes includes.

1. Bacteria.

2. Fungi.

3. Protozoa.

4. Microscopic algae.

It also includes the viruses the non cellular entities sometimes regarded as

being at the border between life and non life.

ESCHERICHIA COLI:

Classification:-

Domain : Bacteria

Phylum : Proteo Bacteria

Class : Gamma Proteo Bacteria

Order : Entero Bacteriales

Family : Entero Bacteriaceae

Genus : Escherichia

Species : Escherichia Coli

Escherichia Coli:

• The proteins group.


25


• Is the most common cause of GIT, UTI, and gram-negative.

• It is main cause of infantile gastroenteritis, traveller’s diarrhoea and neonatal

meningitis.

Bacteriology:

• E. coli: Belongs to the germs Escherichia of the Coliform group.

• It is gram-negative motile bacillus which grows on ordinary media.

• It ferments lactose, a property that distinguishes it from the two major

intestinal pathogens, Shigella and Salmonella.

• It produces pink colonies on MacConkey agar and yellow colonies on agar

because of lactose fermentation.

• It gives a positively methyl red test and a negative vages proskaur reaction and

citrate test.

Pathogenicity:

Although a normal gut commensal, many strain of E. coli have powerful

toxins and other enteropathgenic mechanism which can be responsible for

diarrhea and other symptoms.

STAPHYLOCOCCUS:

Scientific classification:-

Kingdom – Monora

Phylum – Firmicutes

Class – Bacilli

Order – Bacillales

Family – Staphylococcacea

Genus – Staphylococcus

Species – S. aureus


26


GRAM - POSITIVE COCCI:-

Identification of gram positive cocci is an important part of the work the

clinical microbiologist performs. The most common gram-positive cocci infections

include staphylococcus and streptococcus.

STAPHYLOCOCCUS

The genus staphylococcus comprises approximately 19 species, of which there

all important pathogens for humans – S. aureus, S. epidermidis and sprophyticus.

Staphylococci generally appear in stains as grape – like clusters but they can

also appear singly or in pairs. They are approximately 1 mm in diameter.

Staphylococcus is normal flora on the skin and mucous membrane but can

cause infection under certain circumstances.

S. aureus has been recognized historically as a virulent human pathogen. Its

capacity to cause human disease has not been diminished by the introduction of

antibiotics.

For Eg. Such staphylococci can infect prosthetic or indwelling devices (Eg –

artificial body parts and catheters). S.aureus is recovered from various infections,

including skin lesions, basal wound infections, pneumonia and others. Organisms

from these sites on invade the blood and spread throughout the body organisms.

S. aureus can cause such problems as food poisoning, toxic shock syndrome, gastro

enteritis, pneumonia, endocarditis, osteomyelitis and ear and eye infections.

STAPHYLOCOCCUS AUREUS:

Morphology: They are spherical cocci, approximately in grape like clusters. They

may also be found singly, in pairs and in short chain of three or four cell especially

when examined from liquid culture. They are non-motile, non-sporing. They stain

readily with aniline dyes and are uniformly gram positive.


27


Culture characteristic:

They grow readily on ordinary media within a temperature range of 10-42OC.

the optimum temperature being 37OC and pH 7.4 – 7.6. They are aerobes and

facultative anaerobes.

On nutrient agar, after incubation for 24 hrs. The colonies are large (2-4 mm

diameter), circular, convex, smooth, shiny, opaque and easily emulsifiable. Most

strains produce golden yellow pigment, though some may be while orange or yellow.

On nutrient agar slope, their contluent growth presents a characteristic “oil

paint” appearance.

For determining S. aureus a test called coagulase test is performed S. aureus

produce coagulase enzymes that is able to clot rabbit plasma, differentiating this

staphylococcus species from the rest of the clinically significant staphylococci.

PNEUMOCOCCUS:

Pneumococcus, a gram positive lanceolate diplococcus, formerly classified as

Str.pneumoniae, has been reclassified as Stre.pneumoneae because of its genetic

relatedness to Streptococcus. Pneumococcus differs from other Streptococci chiefly in

its morphology, bile solubility, optachin sensitivity and possession of a specific

polysaccharide capsule. Pneumococci are normally inhabitants of the human upper

respiratory tract. They are the single most prevalent bacterial agent in Pneumonia and

in otitis media in children. They can also cause sinusitis, bronchitis, bacteremia,

meningitis and other infections. Pneumococci were first noticed in 1881 by Pasteur

and Steinberg independently.

Morphology:


28


Pneumococci are typically small 1μ m, slightly elongated cocci, with one end

broad or rounded and other end pointed, presenting a flame shaped or lanceolate

appearance. They occur in pairs, with the broad ends in apposition, the long axis of

the coccus parallel to the line joining the two cocci in a pair. They are capsulated, the

capsule enclosing each pair. The capsules are best seen in material taken directly from

exudates and may be lost on repeated cultivation.

In culture the typical morphology may not be apparent and the cocci are more

rounded, tending to occur in short chains. They are non-motile and non-sporing. They

are readily stained with aniline dyes and are Gram+ve.

Cultural characteristics:

Pneumococci have complex growth requirements and grow only in enriched

media. They are aerobes and facultative anaerobes. The optimum temperature being

370C [range 250-420C] and pH7.8 [range 6.5-8.3] growth is improved by 5-10% CO2.

On blood agar, after incubation for 18 hrs, the colonies are small [0.5-1mm], dome

shaped and glistening with an area of green discoloration around them resembling

colonies of str.viridans.

Under anaerobic conditions, colonies on blood agar are surrounded by a zone of beta

hemolysis due to oxygen liable hemolysin O. In liquid media such as glucose broth,

growth occurs as uniform turbidity. The cocci readily undergo autolysis in cultures

due to the activity of intracellular enzymes. Autolysis is enhanced by bile salts,

sodium lauryl sulphate and other surface active agents. Heat cultures do not undergo

autolysis.


29


KLEBSILLA PNEUMONIAE

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose

fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of

the mouth, skin, and intestines.They are short, plump, straight rods, about 1-2× 0.5-

0.8 mm in size. The capsule is often prominent and can be made out even in Gram

stained smears as holoes around the basilli.

It naturally occurs in the soil and about 30% of strains can fix nitrogen in

anaerobic condition.

HISTORY:

The Danish scientist Hans Christian Gram (1853–1938), developed the

technique now known as Gram staining in 1884 to discriminate between K.

pneumoniae and Streptococcus pneumoniae.

Klebssadsadiella was named after the German bacteriologist Edwin Klebs (1834–

1913). Community-acquired pneumonia caused by Klebsiella pneumoniae may be

called Friedländer's Pneumonia, after Carl Friedländer.

Types:

Their classification has undergone various modifications. They have been

classdsified into three species based on biiochemiocal reactions and into over 80

serotypes based on capsular antigens.

Species are,

i. Klebsiella pneumoniae

ii. Klebsiella ozaenae.

iii. Klebsiellarhinoscleromatis.


30

http://en.wikipedia.org/wiki/Gram-negative

http://en.wikipedia.org/wiki/Motile

http://en.wikipedia.org/wiki/Encapsulated

http://en.wikipedia.org/wiki/Lactose

http://en.wikipedia.org/wiki/Fermentation_%28biochemistry%29

http://en.wikipedia.org/wiki/Facultative_anaerobic

http://en.wikipedia.org/wiki/Bacterium

http://en.wikipedia.org/wiki/Nitrogen_fixation

http://en.wikipedia.org/wiki/Hans_Christian_Gram

http://en.wikipedia.org/wiki/Gram_staining

http://en.wikipedia.org/wiki/Edwin_Klebs

http://en.wikipedia.org/wiki/Carl_Friedl%C3%A4nder


Biochemical Reactions:

It ferments sugars (glucose, lactose, sucrose, and mannitol) with the

production of acid and abundant gas. It is indole and MR negative and VP and citrate

positive. Biochemically variant strains are common. It forms Urease. It is second most

populous member of the aerobic bacterial flora of the human intestine. It has become

a very important cause of nosocomial infections.

Pathogenesis:

K. pneumoniae can cause bacterial pneumonia, typically due to aspiration by

alcoholics, though it is more commonly implicated in hospital-acquired urinary tract

and wound infections, particularly in immunocompromised individuals. Klebsiella

ranks second to E. coli for urinary tract infections in older persons. It is also an

opportunistic pathogen for patients with chronic pulmonary disease, enteric

pathogenicity, nasal mucosa atrophy, and rhinoscleroma. Feces are the most

significant source of patient infection, followed by contact with contaminated

instruments.

Members of the Klebsiella genus typically express 2 types of antigens on their

cell surface. The first, O antigen is a lipopolysaccharide of which 9 varieties exist.

The second is K antigen, a capsular polysaccharide with more than 80 varieties. Both

contribute to pathogenicity and form the basis for subtyping.

Klebsiella pneumoniae is a serious disease with high case fatality.It occurs in

middle aged or older persons who have medical problems such as alcoholism, chronic

bronchopulmanary disease or diabetes mellitus.


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http://en.wikipedia.org/wiki/Hospital

http://en.wikipedia.org/wiki/Infection

http://en.wikipedia.org/wiki/Immunodeficiency

http://en.wikipedia.org/wiki/Escherichia_coli

http://en.wikipedia.org/wiki/Opportunistic_infection

http://en.wikipedia.org/wiki/Rhinoscleroma


RESEARCH PROFILE:

• Baldaniya K R,Navasadara nirman ki vidiyon ka proyogika adhyayana evam

stara nirdharana,1979,IPGTR Jamnagar

• Nagendrappa, Management of Kamala with Hareetaki katuki and

Navasadara,1994,GAMC Mysore


32

Methodology

“A comparative Antimicrobial study of Navasadara Satva by taking two different Samples of Navasadara” 33

METHODOLOGY

MATERIALS:

Source of collection of raw material;

The different Samples of chullika lavana were collected from brick

kiln. Three samples were collected from three different brik kilns,

from Koppal, Desur, and Kolhapur and named as sample 1, 2, 3

respectively.

Grahya Navasadara and Khatika were procured from the market,

got authentified by the experts in the subject.

Source of Data:

Literary data is collected from K.L.E.’s B. M. K. Ayurved

Mahavidyalaya Library, K.L.E.’s College of Pharmacy Library,

from well known national journals, and form internet sources.

Navasadara satva was prepared as per the reference, in P.G

Department of KLE’s BMK Ayurvedia Mahavidyalaya,

Belgaum.

Cultures of pathogens, Staphylococcus aureus, E.coli,

S.pneumoniae, K.pneumoniae, are obtained from Maratha

mandal’s Dental College, Microbiology Department in

association with the Microbiologist.

Chullika lavana collection:34

As collection method is not available in any texts of Rasashastra. As

per the reference a material obtained after burning Karira and Pilu kastas in

Methodology


brick kiln is said to be a chullika lavana.. It is available at the bottom of the

brick kiln. Three samples were collected from different places, Koppal, Desur,

and from Kolhapur. Percentage of ammonium chloride in all three samples is

assessed.

4.1 CHULLIKA LAVANA SHODHANA

Equipments:

Tula yantra

Chullika

Vastra

Stainless steel Vessel

Materials with Quantity:

Chullika lavana…. 1 part (600gms)

Jala……………... 3 parts (1800ml)

Procedure:

• Measured quantity of chullika lavana is taken in a stainless steel

vessel

• Measured quantity of jala is poured in the vessel

• Stirred well and kept for some time i.e. 60 min.

• Then it is filtered through a cloth in another vessel.

• This vessel is kept on gas burner to evaporate all liquid part.

• After evaporating all liquid part the white material present at

bottom of the vessel is collected.

Methodology


Observation:

• It is observed that the chullika lavana is not easily dissolved in

water, so it is stirred well and kept for 60 min.

• Room Temperature—300C

• After adding water no changes were observed in temperature.

• Total quantity taken- 600gms

• Qty obtained- 92 gms

• Loss – 508 gms

4.2) NAVASADARA SHODHANA:35

Equipments: Tula yantra

Chullika

Vastra

Stainless steel Vessel

Materials with quantity:

Navasadara…. 1 part (400gms)

Jala……………... 3 parts (1200ml)

Procedure:

• Measured quantity of Navasadara is taken in a stainless steel

vessel

• Measured quantity of jala is poured in the vessel

• Stirred well and kept for some time i.e. 20 min.

• It will dissolve completely.

• Then it is filtered through a cloth in another vessel.

• This vessel is kept on gas burner to evaporate all liquid part.

Methodology


• After evaporating all liquid part the white material present at

bottom of the vessel is collected.

Observation:

• It is observed that the Navasadara is easily dissolved in water,

i.e. within 27 min.

• Immediately water becomes cold.

• Temperature of water before adding Navasadara was 330C, and

after adding Navasadara it was 200C




• Loss – 21 gms

4.3) KHATIKA SHODHANA:36

Equipments: Tulayantra

Khalwa yantra

Stainless steel vessel, spoon/stirrer

Vastra

Measuring jar

Materials with quantity: Raw khatika- 1 part (500gms)

Jala – 4 part (2000ml)

Procedure:

• Measured quantity of khatika is taken in khalwa yantra, finely

powdered.

Methodology


• It is transferred in a vessel containing 4 parts of water.

• Stirred well and filtered through a filter cloth

• Kept undisturbed for 6 hrs

• Then upper water portion is removed

• Khatika present at the bottom of the vessel is dried and collected.

Precaution:

• To observe the separation glass vessel should be used.

Observation:

• Before shodhana khatika is grey in color.

• After mixing water, it easily mixes, and the mixture turns in to

dull grey suspension form.

• After filtering through the cloth, the foreign materials, mud

particles seen on the filtered cloth.

• The filtrate is clean and has the consistency thicker than water.

• For drying, it takes about 2 days.

• Dried shodhita Khatika looks brighter then raw Khatika.




• Loss – 28 gms

Methodology


4.4) NAVASADARA SATVAPATANA (I): 37

Equipments:

Khalwa yantra.

Tulayantra

Damaru yantra

Gopichandan mrittika

Gas burner

Thermo-couple.

Ingredient and Quantity:

Shuddha Navasadara- 4 tola (240 gms) (1tola=12gm=4×12=48=>48×5= 240)

Shuddha Khatika – 3 tola (180 gms) (1tola=12gm=3×12=36=>36×5= 180)

Procedure:

• Preparations of Damaru yantra- 2 equal sized pots were taken. One pot

is placed on another by joining mouth by mouth. Then sandhibandhana

is carried out by using mud smeared cloth.

• Shuddha Navasadara and shuddha khatika is taken in a khalwa yantra

triturated well, until it becomes homogenous mixture.

• Thus formed mixture is placed in Damaru yantra.

• Then this Damaru yantra is kept on gas burner, at a temperature 5500C

for 12hrs.

• Toyadhara is maintained throughout the procedure.

• After swangasheeta is was taken out of gas.

• Sandhibandhana is removed.

Methodology


• Light yellow coloured Satva is collected from the inner part of upper

pot.

Precaution:

• Trituration is carried out until it becomes homogenous mixture.

• Sandhibandhana should be dried completely.

• Toyadhara should be maintained throughout the procedure.

Observation:

• Very less quantity of satva obtained, and more residues remained in

lower pot, so it was triturated once again and subjected for pachana.

Like this procedure is repeated for four times.

• Colour of the residue is pinkish.

• Finally 70gms residue is remained in lower pot.

• Smell of ammonia was observed during pachana.

• Qty taken: 240gms

• Qty obtained: 41.4 gms

4.5) NAVASADARA SATVA (II): 38

Equipments:

Khalwa yantra.

Tulayantra

Damaru yantra

Gas burner

Thermo-couple.

Methodology


Ingredient and Quantity:

Shuddha Navasadara- 4 tola(48 gms) (1tola=12gm=4×12=48)

Shuddha Khatika – 3 tola (36 gms) (1tola=12gm=3×12=36)

Procedure:

• Shuddha chullika lavana and shuddha khatika is taken in a khalwa

yantra triturated well, until it becomes homogenous mixture.

• Thus formed mixture is placed in Damaru yantra.

• Then this Damaru yantra is kept on gas burner, at a temperature 5500C

for 6hrs.

• Toyadhara is maintained throughout the procedure.

• After swangasheeta is was taken out of gas.

• Sandhibandhana is removed.

• Satva was not found.

Precaution:

• Trituration is carried out until it becomes homogenous mixture.

• Sandhibandhana should be dried completely.

• Toyadhara should be maintained throughout the procedure.

Observation:

• Satva not obtained.

• Grey coloured material is found at the bottom pot.

• No smell was observed during pachana.

ANALYTICAL STUDY:

For analytical study samples were labeled as shown below,

Sample A – Raw chullika lavana

Sample B – Shodhita chullika lavana

Methodology


Sample C – Raw Navasadara

Sample D – Shodhita Navasadara

Sample E- Raw Khatika

Sample F- Shodhita khatika

Sample G-Navasadara satvapatana (I)

Sample H-Residue

Sample I-Navasadara satvapatana (II)

PHYSICO CHEMICAL ANALYSIS:39

4.6) Determination of pH value:

Apparatus: pH meter, Glass beaker.

Procedure:

1% of sample solution (1gm sample powder + 100ml of distilled

water) was prepared, Shaken well and homogenized just before taking pH

reading and then the tip of electrode was dipped in such a way that the glass

bulb of the electrode is fully immersed in solution and recorded. In the same

manner pH of all samples were taken out.

4.7) Determination of loss on drying:

Apparatus: Oven, Crucible, and Chemical balance.

Procedure:

1) First evaporating dish is weighed & preconditioned.

2) 2 Gms of sample is taken in pre-weighed, preconditioned

evaporating dish.

3) Then it is placed in hot air oven at 1100 C for half an hour.

4) Difference in the weights of sample before & after placing

in hot air oven is the Loss on drying at 1100C.

Methodology

Calculation:

Wt of the sample – A

Wt of the sample + evaporating dish – B

Wt of evaporating dish + sample after LOD – C

Wt of residue = B-C = D

% Moisture content = D x 100

A

4.8) Determination of Ash value:

Apparatus: Crucible, oven, desiccators, & chemical balance.

Procedure:

1) Crucible is to be conditioned in the oven at 1050 C for one

hour & cooled in Desiccators.

2) Then that crucible is weighed accurately in a chemical

balance.

3) 2 gm of air-dried & powdered sample is taken in conditioned

Crucible.

4) Then it is kept in furnace for incineration at 4500C for 1 hr.

5) % of the total ash content is calculated with reference to the

original weight taken of Air- dried drug for the analysis.

Calculation:

Wt of sample – A

Wt of crucible – B

Wt of crucible after incineration – C


Wt of ash = C – B = D

Methodology

% of Ash value = D × 100

A

4.9) Determination of Acid insoluble ash:

Apparatus: Crucible, Muffle furnace, desiccators, & chemical balance,

beaker.


Procedure:

• After completion of ash value, the ash present in that crucible is

taken for AIA, crucible is washed with 25ml of dilute

Hydrochloric acid, and whole content of that crucible is

transferred into a 100 ml beaker.

• Beaker is kept in water bath for 10 min.

• Then it is filtered through an ash less filter paper.

• Then that filter paper is kept in crucible.

• Crucible is kept in furnace at 4500C for 1 hr.

• Cooled in desiccators, weighed and calculated

Calculation:

Wt of sample = A

Wt of crucible = B

Wt crucible + filter paper after filtering, incinerating = C

AIA = C – B = D

% of AIA = D × 100

A

Methodology

4.10) Specific gravity:40

It is done for 3 samples, raw Navasadara, shodhita Navasadara, and

Navasadara satva (I).

Apparatus: Picnometer, chemical balance.

Procedure: Empty Specific Gravity bottle (picnometer) was weighed and the

bottle filled with distilled water and again weighed, the same bottle was then

filled with 1% (100ml of water + 1gm sample) of sample and weighed. All

these 3 weights were noted; the Specific Gravity of sample was calculated.

Calculation:

Wt of empty picnometer = A

Wt of picnometer + water = B

Wt of picnometer + sample = C

Wt of water = B – A = D

Wt of sample = C – A = E

Specific gravity = E × 1

D

4.11) Solubility:41

Apparatus: Beaker /test tubes, spatula

Procedure: Solubility test of Navasadara satva (I) was subjected with the

following solvents.

Distilled water

Petroleum ether

Alcohol

Chloroform


Methodology


A pinch of sample was taken in a dry test tube with 1ml of solvent and

shaken for 1min. then observed for solubility, non-solubility and sparingly

solubility.

Determination of Elements by ICP-AES Method:

Sample preparation:

0.2500 g of sample was weighed into a Teflon bomb. 8 ml of Milli-Q water,

7 ml of HNO3 and 1 ml of H2O2 was added. The mixture was microwaved for

20 mins in microwave digestor (Model: Milestone Start D). The samples were

then diluted to 50ml with Milli-Q water.Further dilutions may be done if

required to attain the working range of the instrument.

Make of the instrument; Jobin Vyon Horiba

Model: Ultima 2

Procedure:

Normal speed of pump : 20 (rates/min)

Plasma gas flow rate : PL1 (l/min)

Sheath flow rate : G1 (1/min)

Auxiliary flow rate : 0.0 (1/min)

Sheath stabilization time : 15.0 (min)

Nebulisation flow rate : 0.02 (1/min)

Nebulisation pressure : 1.0(bar)

XRD ANALYSIS:42

Definition:

“X-Ray are diffracted because crystalline solids are constructed from a

regularly simple assemblage of components (atoms or atomic groupings)

Methodology


repeated at regular intervals in three dimensions, and X – Ray wavelengths are

the same order of magnitude as the spacing of atom centers.”

From Morphological view-point, a crystal may be defined as the

regular polyhedral form, bounded by smooth surfaces, which is assumed by a

chemical compound under the influences of it’s inter atomic forces when

passing from the gaseous or liquid state to solid state.

The geometric shapes of crystals reflect an internal symmetry of atoms

and molecules arranged in a regular and repeated pattern in space, which

distinguish crystalline solids from amorphous.

An important aspect of solid state is the ability of compounds to

crystallize in a variety of symmetrical arrangements of their molecules in

space i.e. polymorphic forms. Which are often quite different from each other

in physical characters (Habits, Melting point, solubility) although chemically

identical.

The pharmaceutical relevance of polymorphisms lies in the fact that

the differences in physico-chemical properties between forms may influence

manufacturing processes and importantly, dissolution rate and therefore

bioavailability.

The X – Ray diffraction methods are necessary to distinguish between

different crystallographic arrangements.

The condition for diffraction of a beam of X – Ray from crystal is

given by Bragg’s equation:

2 d Sin θ = n λ

Where,

λ = Wavelength of X – Ray beam

Methodology


θ = Angle between incidents

X-Ray and crystal lattice plan

d = Distance between lattice plane’s

The Bragg’s equation is fundamental relation underlying all X-Ray

Diffraction measurements.

Only for angles of incidence such as Sin θ = n λ / 2 d will W – Rays be

reflected, and at all other angles destructive interference occurs.

The specimens can be identified by reference libraries such as Powder

Date File or the Joint Committee on Powder Diffraction Standards (JCPDS),

which contains the diffraction patterns of some tens of thousands of

substances in numerical form.

Advantages:

• The particular advantage of diffraction analysis is that it discloses the

presence of a substance as that actually exists in the sample, and not in

terms of its constituent chemical elements. i.e. this analysis shows both

the elemental form and compound form of the element as it is present

in the sample.

• Helps to identify and characterize the raw materials of mineral/metal

origin.

• It is also used to fix up the particular characteristics pattern (finger

print) of prepared bhasma.

Methodology


4.12) Qualitative analysis of samples:43

Calcium:

Dissolve 0.8gm of sample in 20 ml con HCl => boil the solution =>

filter => collect the filtrate and test.

Test: Filtrate + ammonia+ potassium ferocynide => yellow ppt => calcium

present

Sodium / Potassium / Chlorides:

Dissolve 2 gm of sample in 20 ml conc. HCl => boil the solution

=>filter through filter paper => collect the filterate and test.

Test: Filtrate + Potassium Pyroantimonate => Milkyness ppt => Sodium

present.

Filtrate + Sodium cobalt nitrate => Yellow ppt => Potassium present.

Filtrate + Silver nitrate solution => White ppt => Chlorides present.

Ammonia:

Sample + NaOH => boil and hold a wet litmus paper at the mouth of

the test tube.

Observation:

Gas with ammonia smell having alkaline action on litmus paper (red

turns to blue) and white dense fumes with glass rod dipped in concentrated

HCl.

4.13) Antimicrobial study:44

Aim of the study

In the present study, Disc diffusion / cup plate method and Broth

dilution both tests are performed to determine the level of bacteriostatic

and bactericidal concentration.

Methodology


4 samples were selected for antimicrobial study they are,

shodhita chullika lavana, shodhita Navasadara, Navasadara satva (I) and

Navasadara satva (II), they are labeled as sample 1, 2, 3, 4 respectively.

Methods:

The antimicrobial study was designed by following two methods as;

1. Agar disc diffusion method

2. Broth dilution method.

1) Agar disc diffusion method

This is one of the methods officials in Indian Pharmacopeia. Where

the Antimicrobial powder dissolved in saline water is diffused from the

cup through an agar layer in petri-dish to an extent such that the growth

of added microorganisms is restricted entirely in circular area or zone

around the cavity containing the solution of an antibiotic substance.

The Antimicrobial activity is expressed as zone diameter in

millimeters, which is measured with a divider.

Samples were screened for Antimicrobial activity against micro-

organisms and the activity was compared with appropriate standard

allopathic drugs.

Antimicrobial activity:

The Antimicrobial activity of a drug is generally expressed as its

inhibiting effect towards the growth of bacterium in nutrient broth or

nutrient agar.

For this study following conditions are required:

o The substance must be contact with the test organism.

Methodology


o Condition must be favorable for the growth of

microorganisms in the absence of Antimicrobial substances.

o There must be a means of estimating the amount of growth

and thereby, percentage of growth of inhibition.

o The activity of powder substance should be observed and

determined by the growth response of microorganisms.

Materials used:

o BHI Agar media

o Stock solution

o Inoculated Microorganisms solution

o Sterile micro-pipettes

o Petri dishes

o Sterile (metal)borer

o Cotton swab

o Incubator

o Spirit lamp

o Micropipettes

Standards used in the study:

o Ciprofloxacin 2mg/ml

Preparation of stock solution:

1. 500 mg of all 4 samples are taken and dissolved in 1 ml of

saline water to get a concentration of 500mg/ml.

2. From prepared stock solution main test solutions are

prepared by taking different four concentrations i.e.

Methodology


1:2,1:5,1:10, & 1:20 and tested for its antibacterial activity,

totally four concentrations were tested.

Preparation of test solution:

The test solution here prepared using saline water to get the

following concentrations

1. 1:2 -------- 125 mg/ml

2. 1:5 ------- 62.5 mg/ml

3. 1:10 ------- 13mg/ml

4. 1:20 ------- 6mg/ml

Preparation of inoculums:

Mueller - Hinton agar medium (Himedia labs) of the following

composition was used for preparation of slants.

Peptone ---- 5.0 g

Beef extract ---- 1.5 g

Sodium chloride ---- 5.0 g

Agar ---- 15.0 g

Yeast extract ---- 15.0 g

Distilled water to make ----- 1000 ml

• About 28 gm of prepared medium was taken in 1000 ml of

distilled water and boiled to dissolve completely. After that

streaked with micro organisms.

• The slants were incubated at 37OC + 1OC for 24 hrs.

• These 24 hrs cultures were used for preparation of inoculums.

Methodology


• The suspension of micro organisms was prepared in 10 ml of

sterile water and 0.5 ml of this suspense was added for 10 ml of

the agar medium.

Culture Medium:

Different types of media have been used according to the types of

organisms.

In the present study, BHI (Brain heart infusion) agar media employed

possessing the following composition (Ready made medium).

Agar ---- 15.0 g

Calf brain infusion from ---- 200.00

Beef heart infusion from ---- 250.00

Proteose peptone ---- 10.00

Dextrose ---- 2.00

Sodium chloride ---- 5.00

Disodium phosphate ---- 2.50

About 52.0 gm of above readymade medium was dissolved in

distilled water (1000ml) by gentle heating. Heat to boiling to dissolve

the medium completely. Sterilize by autoclaving at 15lbs pressure

(1210C) for 15mins. Mix well before pouring.

Sterilization:

Sterilization of the medium, tubes for slants, borer etc. was done

by autoclaving at 15 ibs / square inch for 20 mins. The glass wares like

syringes, petridishes, pipettes, empty test tubes, were sterilized by dry

heat in an oven at a temperature of 160OC for 1 hrs.

Methodology


Preparation of Agar plates:

The sterilized medium was cooled at 40OC and 0.5 ml of

inoculums per 100 ml of medium was added to the conical flask.

This was shaken gently to avoid the formation of air bubbles and then

transferred into petri-dishes. So as to obtain 6mm thickness of medium.

The medium in the plate was allowed to solidify at room temperature.

Thus prepared agar used for three organisms except S.pneumoniae

Readymade Chocolate agar was used for S.pneumoniae.

Experimental Procedure:

• The sterile borer was used to prepare five cups of 8 mm diameter

in the medium of each petri-dish.

• Accurately measured 50 mL solutions of each concentration of

solutions of dissolved powder (prepared standard drug) were

added to the cups with the help of micropipette.

• All the plates were kept at room temperature for effecting

diffusion of drug dissolved and standards.

• Later, they were incubated at 37+1OC for 24 hrs.

• The presence of definite zones around the cup of any size

indicated antibacterial activity.

• The control was run simultaneously to assess the activity of

saline water which was used as the vehicle for drug powder.

• The diameter of the zone of inhibition was measured and

recorded.

Methodology


• The zones of inhibition for the antibacterial activities of samples

were calculated by measuring the inhibitory effect towards the

growth of bacteria around nutrient agar cup.

1) Broth dilution test Method:

The Broth Dilution method is a simple procedure for testing a

small number of isolates, even single isolate. It has the added advantage

that the same tubes can be taken for MBC tests also:

Materials:

o B H I Broth media

o Overnight broth culture of test and control organisms

(Inoculated microorganisms)

o Samples (required antibiotic in powder form).

o Required solvent- sterile Distilled Water.

o Sterile capped 7.5 x 1.3 cm tubes / small screw-capped

bottles,

o Incubator

o Cotton swab

o Sterile graduated Micropipettes-5, 10, 50, 200, 500mL size.

o A suitable rack to hold 40 tubes in four rows i.e. 10 tubes in

each row.

Preparation of Broth media:

Brain Heart Infusion media was used for the preparation of Broth

solution.

Methodology


Calf brain infusion from ---- 200.00

Beef heart infusion from ---- 250.00

Proteose peptone ---- 10.00

Dextrose ---- 2.00

Sodium chloride ---- 5.00

Disodium phosphate ---- 2.50

Suspend 37.0gms in 1000ml distilled water dispense in to bottles

or tubes as desired. Sterile by autoclaving at 15lbs pressure (1210C).

Preparation of Stock solution:

• Stock solution is prepared by using saline water.

• The stock solution is prepared in the concentration 1gm/ml. The

volume of the solution used during the procedure is 0.2mL, so

the concentration of the drug finally stands as 1mg/ml.

Experimental procedure:

• To start with, glass test tubes plugged with cotton all are

autoclaved.

• Ten Borosilicate glass test tubes are taken in four rows each, one

corresponding for the serial dilution w.r.t one organism.

• Test tubes are serially numbered from 1-10.

• The first test tube placed in the row in rack with the name of the

organism used in the study.

• 200 mL of the (BHI) Brain heart infusion broth is initially taken

in all the test tubes of eight rows.

• To the first tube of all the four rows, 200mL of the stock solution

is added.

Methodology


• It is pipette in and out to ensure uniform mixing.

• 200 mL of the first test tube is then pipette to the second tube and

the process is repeated till the 10th tube.

• From the 10th tube 200 mL is pipette out and discarded.

• Every time when 200 mL of solution is introduced in the test

tube it is gently pipette in and out within the test tube to ensure

proper mixing.

• During every withdrawal of 200 mL of the solution and

introduction to another tube, a new pipette is made use of.

• In the above said method, serial dilutions are made similarly with

10 test tubes arranged in sixteen rows corresponding for four

microorganisms of four drug samples.

• The concentration in the test tube now stands as 250mg (tube-1),

125mg(tube-2),62.5mg(tube-3),31.5mg(tube-4),16mg(tube-5),

8mg (tube-6), 4mg(tube-7), 2mg(tube-8), 1mg(tube-9), 0.5mg

(tube 10)

• Now adjust the bacterial count 1.58*108 organisms per ml by

comparing with McFarland’s standards. The suspension of the

organisms, , Staphylococcus aureus, E.coli, K.pneumoniae,

S.pneumoniae in the ( BHI) Brain heart infusion Broth is then

inoculated to all the test tubes of a corresponding four rows ,in

the concentration of 200 MicroLitres.

• All tubes are inoculated at 350C for 24hrs without shaking or

agitation.

Methodology


• After 24hrs of incubation the test tubes are read for MIC is

defined as the concentration of the first tube in the series to show

visible traces of growth of bacteria, which is evident, by turbidity

or cloudiness of the solution in the test tube.

• Turbidity indicates that bacterial growth has not been inhibited

by the concentration of the preparation contained in the medium.

• The main advantage of the ‘Broth dilution’ method for the MIC

determination lies in the fact that it can readily be converted to

determine the MBC as well.

Reading of result:

• MIC is expressed as the lowest dilution, which inhibited growth judged

by lack of turbidity in the tube. Because very faint turbidity may be

given by the inoculums itself, the inoculated tube kept in the

refrigerator overnight may be used as the standard for the

determination of complete inhibition.

Minimum Bactericidal Concentrations (MBC):

• The MBC values also known as MLC (Minimal lethal

concentration) is the estimation of bacterial activity and is

defined as the lowest concentration of the preparation of the

sample to kill 99.9% of the initial inoculums after incubation for

24hrs under a standardized set of conditions. By observing MIC

result 3rd sample (Navasadara Satva I) was taken for MBC. Here

in the present study MBC was obtained by platting out the

contents on an agar medium to count the growth.

Methodology


Method

• Dilutions and inoculations are prepared in the same manner as

described for the determination of MIC or the dilutions prepared

for MIC can be taken directly for MBC.

• Next day readings are taken, after visually clear tubes 0.1ml from

each of four tubes were taken i.e. 2 sensitive and 2 turbid &

spread across the surface of dried agar plates with the help of

sterile bent glass rods. The plates were inoculated overnight at

350C for 24hrs.

• Next day plates are observed for growth of colonies and readings

are noted. The number of colonies that grown on each MBC

subculture plates after overnight incubation was counted.

• The concentration in which petriplate showed either no growth or

reduction in 99.9%, was considered the MBC of specific

antimicrobial agent.

Reading of result

• These subcultures may show similar number of colonies-

indicating bacteriostasis only.

• A reduced number of colonies-indicating a partial or slow

bactericidal activity.

• No growth- if the whole inoculum has been killed.

• The highest dilution showing at least 99% inhibition is taken as

MBC.

Results

RESULTS 5.1 Observational Results: The observation which has been done during the study has shown below.

Table NO.6-Organoleptic characters of raw materials:

SAMPLES ORGANOLEPTIC CHARACTERS

COLOUR TOUCH ODOUR TASTE

1 Grey Rough Muddy Muddy

2 White Soft Burnt Ash

3 Brown Soft Muddy Muddy

“A comparative Antimicrobial study of Navasadara Satva by taking two different samples

of Navasadara”

59

Table No.7-Chullika lavana shodhana:

CHULLIKA

LAVANA

ORGANOLEPTIC CHARACTERS


Before shodhana Whitish soft odourless Corrosive

After shodhana Whitish soft odourless Corrosive

Table No.8-Navasadara shodhana:

NAVASADARA ORGANOLEPTIC CHARACTERS


Before shodhana Whitish crystal odourless Corrosive

After shodhana Whitish crystal odourless Corrosive

Results

Table No.9- Khatika shodhana:

KHATIKA ORGANOLEPTIC CHARACTERS


Before shodhana Dull Grey soft Characteristic Tasteless

After shodhana Bright Grey soft Characteristic Tasteless


of Navasadara”

60

Table No.10-Navasadara satvapatana (I):

NAVASADARA

SATVA (I)



Yellow soft characteristic Alkaline

odour of ammonia

Table No.11-Navasadara satvapatana (II):

NAVASADARA

SATVA(2)



Grey soft Odourless Tasteless

5.2 ANALYTICAL RESULTS: Table No.12-Raw material analysis: Sr. No. Parameters Sample 1 Sample 2 Sample 3

1 % of Ammonia 2.34% 0.82% 0.64%

2 % of Chloride 1.36% 14.68% 1.90%

3 % of Calcium 2.20% 40.67% 1.48%

Results


of Navasadara”

61

4 % of Magnesium 0.48% 2.68% 1.80%

5 % of Potassium 0.66% 0.38% 0.26%

6 % of Iron 1.49% 0.76% 2.94%

7 % of Silica 90.16% 9.88% 61.94%

9 % of NH4Cl 2.01% 2.32% 1.054%

Table No.13-Physico-chemical parameters: SAMPLE pH LOD ASH

VALUE AIA

SPECIFIC GRAVITY

A 11.50 1.84%w/w 84.47% 17.56% NA

B 12.43 7.11%w/w 81.47% 11.38% NA

C 7.8 13.23%w/w 0.046% NIL 1.0045

D 8.0 15.86%w/w 87.83% NIL 1.0046

E 13.0 2.63%w/w 80.15% 65.62% NA

F 9.0 4.15%w/w 77.98% 22.24% NA

G 10.1 14%w/w 0.099% NIL 1.0046

Table no. 14- Solubility: 01 Petroleum ether + Navasadara Satva(I) Insoluble

02 Alcohol + Navasadara Satva(I) Sparingly soluble

03 Chloroform + Navasadara Satva(I) Insoluble

04 Water + Navasadara Satva(I) Soluble

Results

Table No.15-Qualitative Analysis:

Sl.no. Parameter SAMPLES

A B C D E F G

01 Ammonia Absent Absent Present Present Absent Absent Present

02 Sodium Present Present Absent Absent Present Present Present03 Potassium Absent Absent Present Present Absent Absent Absent 04 Chloride Present Present Present Present Present Present Present

05 Calcium Absent Absent Absent Absent Present Present Absent

Table No.16-Quantitative Analysis:

Sample % of

NH3

%

NH4Cl

% of Cl % of Ca Mg K Fe Silica

A 3.64% 64.24% 39.24% NA +NT +NT +NT 12.61%

B 8.96% 60.70% 36.90% NA +NT +NT +NT 16.34%

C 7.34% 95.70% 58.18% NA +NT +NT +NT 0.27%

D 2.59% 90.82% 55.49% NA +NT +NT +NT 0.082%

E --- --- --- 78.90% +NT +NT +NT 0.098%

F --- --- --- 76.60% +NT +NT +NT 0.11%

G 3.68% 79.01% 48.65% 0.38% +NT +NT +NT 1.73%

H 0.98% 54.29% 32.88% 3.67% +NT +NT +NT 0.22%

I 1.09% 68.94% 41.87% 1.82% +NT +NT +NT 0.64%


of Navasadara”

62

Results

Table No.17-AES-ICP Result of the sample Navasadara Satva (I):

Navasadar satva(I) In ppm Si 80.3 Ca 166.7 Fe 925.4 K 31.8 Mg 54.9

XRD result of Navasadara Satva (I):

Peak search report NH4Cl

PEAK: 29-pts/parabolic filter, threshold=2.0, cutoff=1.5%, BG=7/1.0, Peak-

Top=summit

Intensity = counts, 2T (0) = 0.0(0), Wavelength to compute d-Spacing cubic =

1.54056A (Cu/K-alph…)

5.3 ANTIBACTERIAL SCREENING RESULTS:

Table No. 18- Results of cup-plate diffusion method

Zone of inhibition in mm :

SAMPLE

Std Cipro

Dilutions of samples in different concentrations

ORGANISMS

1 2 3 4 > 40

S.aureus

18 R 26 R > 40

S.pneumoniae

R R 29 R > 38

E.coli

20 R 32 14mm > 40

Klebisella pneumonia

14 R 18 R > 40

1:2

S.aureus

R R 16 R > 40

1:5 S.pneumoniae

R R 15 R > 36


of Navasadara”

63

Results

E.coli

17 R 16 R > 40


12 R 14 R > 40

S.aureus

20 R 14 R > 40


of Navasadara”

64

S.pneumoniae

R R R R > 36

E.coli

R R 15 R > 40


R R R R > 38

1:10

S.aureus

R R R R > 40

S.pneumoniae

R R R R > 40

E.coli

R R 12 R > 40

1:20


R R R R > 36

Results

Table No.19-Results of Macrodilution broth susceptibility method:

Sample Micro

Organisms

250

mg/ml

125

mg/ml

62.5

mg/ml

31.25

mg/ml

16

mg/ml

8

mg/ml

4

mg/ml

2

mg/ml

1

mg/ml

0.5

mg/

ml

S.aureus S S S S R R R R R R

S.pneumoniae

S S S S S R R R R R

E.coli R R R R R R R R R R

1


S S S S R R R R R R

S.aureus S S S S R R R R R R

S.pneumoniae

S S R R R R R R R R

E.coli R R R R R R R R R R

2


S S R R R R R R R R

S.aureus S S S S S S R R R R

S.pneumoniae

S S S S S R R R R R

E.coli S S S S R R R R R R

3


S S S S S R R R R R

S.aureus S S R R R R R R R R

S.pneumoniae

S R R R R R R R R R

E.coli R


of Navasadara”

65

R R R R R R R R R

4


S R R R R R R R R R

Results


of Navasadara”

66

Table No.20-Result of MBC method

Minimum bactericidal concentration of Sample 3:

ORGANISM

CONCENTRATION (in mg)

RESULT

16.125 34 colonies 8.00 78 colonies 4 .00 More than 100 colonies

Staph aureus

2 .00 More than 1000 colonies

31.250 No Growth 16.125 70 colonies 8.00 More than 1000

colonies

Staphylococcus pneumonia

4.00 More than 1000 colonies

31.250 More than 1000

colonies 16.125 More than 1000

colonies 8.00 More than 1000

colonies

Klebsiella .pneumoniae

4.00 More than 1000 colonies

62.5 No Growth 31.250 14 colonies 16.125 No Growth

E coli

8.00 One colony

Conclusion

CONCLUSION

• Chullika lavana couldn’t yield satva.

• Even though chullika lavana is a source of Navasadara, it showed less % of

ammonium chloride and also it is less effective when compared to market

sample of Navasadara.

• Shodhita chullika lavana, shodhita navasadara and satva (II) showed

bacteriostatic activity against all organisms except E coli.

• Navasadara Satva (I) showed bacteriostatic activity on all four organisms.

• Navasadara Satva (I) prepared by using market sample of Navasadara) is said

to be a effective drug against the organisms S.pneumoniae and E coli, at a

concentration of 31.250mg and 62.5mg respectively, at this stage it exhibited

bactericidal effect.

• Artificially prepared Ammonium Chloride can be used in place of Navasadara.

• Ammonium chloride prepared artificially has satisfied properties mentioned in

classics.

• Compared to shodhita Navasadara, Navasadara Satva is more effective.

• Preparation of Navasadara satva is less laborious and also cost effective.

70 “A comparative Antimicrobial study of Navasadara Satva by taking two different samples of

Navasadara”

Discussion

“A comparative Antimicrobial study of Navasadara Satva by taking two different Samples of Navasadara”

67

DISCUSSION

Whole work can be discussed under following headings

Discussion on raw material collection

Discussion on Preparation

Discussion on Analytical part

Discussion on Antimicrobial part

• As per the classics Navasadara is extracted from chullika lavana that to

particular plants should be used as a fuel, i.e Karira (Capparis decidua

Edgew), and Pilu (Salvadora persica Linn), and now a day these drugs are not

used as fuel in brick kiln, so less quantity of Navasadara is found.

• The white part which is remained after burning brick in kiln is also said to be

ishtika lavana or chullika lavana or Navasadara. Now a day in brick kiln

nimba kasta is used as a fuel, and in some kilns coal is used, so required

amount of Navasadara is not extracted from chullika lavana.

• All three samples of chullika lavana differ in colour, it might be because of the

things used as a fuel in that brick kiln.

• Pattern of agni is not mentioned for satvapatana, only for Navasadara

shodhana teevragni is mentioned. More amount of agni is required for

satvapatana i.e. 12 hrs pachana has been done to get Satva. It is fixed after

doing pilot study.

• Chullika lavana couldn’t yield Satva; it might be because of the very less

percentage of ammonium chloride in it.

• Compared to Navasadara, pH of chullika lavana is high i.e. more alkaline

when compared to Navasadara.

Discussion


68

• LOD is maximum in raw Navasadara, shodhita Navasadara and Navasadara

Satva because of more amount of moisture content in it.

• Acid insoluble ash of raw Navasadara, shunavasadara and Satva is nil because

it will not sustain 4500 C temperatures i.e. it evaporated.

• No significant difference observed in specific gravity of the samples C,D&G.

• % of ammonia in Navasadara Satva (I) is 3.68%, in Navasadara Satva (II) it

was 1.09%, and in residue it was 0.98%.

• After extraction of Satva also some amount of ammonia is found in residue.

• ICP-AES results of Navasadara Satva (I) reveals presence of elements like Si,

Ca, Fe, K, Mg in ppm level of in the in ppm level.

• XRD showed highest peak of NH4Cl, it showed presence of ammonium

chloride in the sample Navasadara Satva (I), and it cubic in structure.

• For antimicrobial study 4 samples were taken to assess the activity, they are

shodhita chullika lavana, shodhita Navasadara, Navasadara satva (I) and

Navasadara satva (II) and they are labeled as sample 1, 2, 3, 4 respectively.

• Antimicrobial study revealed,

Minimum inhibition concentration (MIC):

Sample 1- Shodita chullika lavana

• E-coli showed resistance.

• Sensitivity at a concentration of 31.250mg/ml onwards for k pneumoniae &

s. aureus.

• S.pneumoniae showed sensitivity at a concentration of 16.125mg/ml onwards.

Sample 2- Shodhita Navasadara

• E-coli showed resistance.

Discussion


69

• S aureus showed sensitivity at 31.250mg/ml

• S.pneumoniae & K pneumoniae showed sensitivity at 125mg/ml onwards

Sample 3-Navasadara Satva (I)

• E coli showed sensitivity at 31.125mg/ml

• S aureus showed sensitivity at 8mg/ml onwards

• S pneumoniae & k pneumoniae showed sensitivity at 16.125mg/ml

Sample 4-Navasadara Satva (II)

• E coli showed resistance S aureus sensitive at 125mg/ml S pneumoniae & k

pneumoniae sensitive at 250mg/ml onwards.

• Sample 1, 2 showed sensitivity in all organisms except E-coli, it might be

because of its high pH level i.e. alkaline property.

• Among all 4 samples, 3rd sample showed good bacteriostatic effect, so it was

selected for MBC to evaluate its bactericidal effect.

• MBC- sample 3 exhibited bactericidal effect for S.pneumoniae at a

concentration of 31.250mg and for E coli it was 62.5 mg, means at this level

of concentration no growth was found.

• Disc diffusion - at 1:2 & 1:20 concentration 3rd sample showed maximum

zone of inhibition against E-coli. At 1:10 & 1:5 concentration sample 1

showed maximum zone of inhibition against S.aureus and E coli respectively.

• Hence disc diffusion method was adopted by comparing zone of inhibition

between test drugs against control drug i.e. ciprofloxacin. It was found that

drug has shown antimicrobial result against S.pneumoniae and E-coli

compared to other organisms.

Summary


72

SUMMARY

Whole work is summarized as follows-

Navasadara satvapatana is only mentioned in the text Rasatarangini. It is

indicated in Puppusashotha, Shweat kutsa, Amlapitta, Murcha, Kaphanissaraka etc. so

this study has been conducted to screen antimicrobial activity of the Navasadara

satva. It is said that we can extract Navasadara from chullika lavana. So here an effort

has been taken to extract Navasadara from chullika lavana. The present study

conducted to assess the physico-chemical features of chullika lavana, Navasadara and

Navasadara Satva and its anti microbial activity on two gram positive and two gram

negative organisms.

For the study raw materials like Navasadara and khatika were collected from

market and chullika lavana is collected from three different brick kiln, i.e. koppal,

Desur, and from Kolhapur. After analysis, on the base of % of Ammonium chloride

one sample of chullika lavana i.e. Desur sample is selected for study.

Chullika lavana, Navasadara and Khatika are subjected for shodhana process

by nirmalikarana. Navasadara Satva is prepared by doing pachana in damaru yantra.

Navasadara satvapatana is carried out as per the reference Rasatarangini. Got yellow

coloured Satva from the market sample Navasadara, whereas from chullika lavana

Satva was not found. It might be because of the less % of Ammonium chloride in it.

The physico–chemical analysis of Chullika lavana, Navasadara and khatika

before and after shodhana and Satva has been carried out to evaluate their qualitative

and quantitative assessment. Final product contains maximum quantity of ammonium

chloride i.e. 79.01%, remaining are trace elements such as, Calcium, Magnesium,

Silica.

Summary


72

For Antimicrobial study four samples were selected, they are Shodhita

chullika lavana, Shodhita Navasadara, Navasadara Satva (I), and Navasadara Satva

(II), labeled as sample 1, 2, 3, 4 respectively.

Antimicrobial study was carried on four different strains of Micro-

organisms by Agar disc diffusion method compared with standard drug with the four,

and Minimal inhibitory concentration method for the more accuracy of the study.

After the study it is revealed that the Navasadara Satva (I) shown good

sensitivity on micro-organisms. All four samples showed bacteriostatic effect on all

organisms except E-coli. Whereas 3rd sample showed bacteriostatic effect on all

organisms. So the 3rd sample is selected for MBC to assess its bactericidal effect. 3rd

sample is said to be a good drug against the organisms S.pneumoniae and E coli, at a

concentration of 31.250mg and 62.5mg respectively, at this stage it exhibited

bactericidal effect.

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76


Photos

RAW MATERIALS

COLLECTION OF CHULLIKA LAVANA FROM BRICK KILN

KHATIKA NAVASADARA

PLATE NO 1:NAVASADARA AND KHATIKA

A comparative Antimicrobial study of Navasadara Satva by taking two Different Samples of Navasadara

Photos

NAVASADARA CHURNA NAVASADARA

FILTERATE

FILTERATION

FILTERATE KEPT ONBOILING

SHODHITA NAVASADARA SHODHITA NAVASADARA CHURNA


Photos

NAVASADARA SHODHANA PLATE NO 2:

CHULLIKA LAVANA CHULLIKA LAVANA + JALA

RESIDUE KEPT ON AGNI


Photos

SHODHITA CHULLIKA LAVANA

PLATE NO-3: CHULLIKA LAVANA SHODHANA

RAW KHATIKA POWDERING RAW KHATIKA+JALA


Photos

FILTERING AFTER 6 HRSKEPT UNDISTURBED

SHODHITA KHATIKA KEPT FOR DRYING

PLATE NO-4: KHATIKA SHODHANA


Photos

SHU.NAVASADARA +KHATIKA MIXING

KEPT IN POT SANDHIBANDHANA PACHANA

LOWER POT RESIDUE UPPER POT SATVA

PLATE NO-5: NAVASADARA SATVAPATANA (I)


Photos

SHU.CHULLIKA LAVANA & KHATIKA MIXING


KEPT IN POT PACHANA

Photos

SATVA (II)

PLATE NO-6: NAVASADARA SATVA (II)

MICROPIPETTE BHI BROTH


Photos

INCUBATION OF PLATES INCUBATOR

INCUBATION OF TUBES

PLATE NO-7: EQUIPMENTS USED IN ANTIMICROBIAL STUDY

S.AUREUS S.PNEUMONIAE

250 125 62.5 31.25 16 8 4 2 1 0.5mg

SAMPLE-3 SAMPLE-3


Photos

K.PNEUMONIAE E.COLI

SAMPLE-3 SAMPLE-3

PLATE NO-8: ANTIMICROBIAL STUDY -MIC METHOD

E.COLI 4 SAMPLES


Photos

32mm

Zone of Inhibition

S.AUREUS 4 SAMPLES:

PLATE NO-9: ANTIMICROBIAL STUDY- DISC DIFFUSION METHOD

K.PNEUMONIAE 4 SAMPLES:


Photos


S.PNEUMONIAE 4 SAMPLES


Photos



Photos

SAMPLE -3

PLATE NO-12: ANTIMICROBIAL STUDY- MBC METHOD


“a comparative antimicrobial study of navasadara …

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