PHARMACEUTICO - ANALYTICAL AND TOXICOLOGICAL STUDY OF SHUDDHA MALLA

ON WISTAR ALBINO RATS (AN EXPERIMENTAL STUDY)

BY

DR. K. PALLAVIDR. K. PALLAVIDR. K. PALLAVIDR. K. PALLAVI

Dissertation submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, KARNATAKA, BANGALORE.

In partial fulfillment of the requirements for the degree of

AYURVEDA VACHASPATI (DOCTOR OF MEDICINE)

In

RASASHASTRA

Under The Guidance of

Dr.SHANKAR GOWDA M.D.(AYU)

Asst.Professor T.G.A.M.C., Bellary .

DEPARTMENT OF POST GRADUATE STUDIES IN

RASASHASTRA

TARANATH GOVT. AYURVEDIC MEDICAL COLLEGE, BELLARY – 583 101

(KARNATAKA) 2008

2

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, KARNATAKA, BANGALORE.

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled :

“Pharmaceutico – Analytical and toxicological Study of

Shuddha Malla on Wistar albino rats (An Experimental

Study)” is a bonafide and genuine research work carried out by me

under the guidance of Dr.Shankara Gowda, M.D. (Ayu),

Asst.Professor, T.G.A.M.C, Bellary.

Signature of the Candidate

Date : DR. K.PALLAVI

Place : Post Graduate Scholar in Rasashastra

T.G.A.M.C, Bellary.

3

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, KARNATAKA , BANGALORE.

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled:

“Pharmaceutico - Analytical and toxicological Study of

Shuddha Malla on Wistar albino Rats -An Experimental

Study” is a bonafide research work done by DR.K.PALLAVI in

partial fulfillment of the requirement for the degree of Ayurveda

Vachaspati, Doctor of Medicine (Ayurveda).

Signature of the Guide

Date : Dr.SHANKARA GOWDA, M.D. (Ayu)

Place : Asst. Professor

T.G.A.M.C., Bellary.

4

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, KARNATAKA, BANGALORE.

ENDORSEMENT BY THE HOD, PRINCIPAL/

HEAD OF THE INSTITUTION

This is to certify that the dissertation entitled

“Pharmaceutico analytical and toxicological Study of Shuddha

Malla on Wistar Albino Rats-An Experimental Study” is a

bonafide research work done by DR.K.PALLAVI under the

guidance of Dr.Shankara Gowda, M.D. (Ayu) Professor, T.G.A.M.C.

Bellary.

Seal & Signature of the H.O.D Seal & Signature of the Principal

Dr. M.S.DODDAMANI, M.D. (Ayu) Dr. K.VISHWAMBHARA M.D.(Ayu)

Date : Date :

Place : Place :

5

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCES, BANGALORE.

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health

Science, Karnataka shall have the rights to preserve use and

disseminate this dissertation in print or electronic format for

academic / research purpose.

Signature of the candidate

Date :

DR.K.PALLAVI

Place:

6

ACKNOWLEDGEMENT

I pray and bow before almighty for having given me this unique opportunity.

It gives me pleasure to express my gratitude with profound respect to my guide

Dr.Shankara Gowda, Asst.Professor, Dept of PG Studies in Rasashastra, T.G.A.M.C.,

Bellary for his scholarly guidance, constant encouragement throughout my study.

I am overwhelmingly grateful to Dr.M.S.Doddamani, Professor& HOD, Dept of

Post Graduate Studies in Rasashastra, T.G.A.M.C., Bellary for his moral encouragement,

inspiration and continuous support in completing my work.

I am blessed to have precious supervision, pinpoint suggestion and constant help

of Dr.N.B.Shridhar,MVSc,Phd Asst.Professor,Principal Investigator, Department of

Pharmacology & toxicology, KVAFSU, who guided me during experimental study.

I extend my gratefulness to honourable Principal Prof. K.Viswambhara M.D (Ayu)

for providing all facilities to make the study success.

I express my profound sense of gratitude to all the PG Scientific committee

members Dr. Shobha Hiremath,Dr.Surekha Madikeri & Dr.Ravi Chavan for their

continuous help & suggestions.

I am extremely grateful to the respected & esteemed ,Dr.Sathyanarayana Bhat,

Dr.G.R.Vastrad, Dr.L.N. Kundargi, Dr. V.L. Yadhalli, Dr. Hugar, Dr. Saraswathi,

Dr.Shashikala Biradar, Dr. Rajshekar Ganiger, Dr. Srivatsa,Dr.Madhava Diggavi , for

their inspiration and encouragement.

I am cordially thankful to Prof.Subodh ,Prof.Subramanian ,Prof.Deshpande,

Mr.Krishnamurthy, Dept.of Materials Engineering, Indian Institute of Science,Bangalore

and Ganesh Consultancy & Analytical Services for their kind co-operation in performing

analytical study.

My Special thanks to Prof.Narayan Swamy, Dept of Pathology, KVAFSU for his

kind co-operation in histopathological study.

I am thankful to Mr.Srinivas, Vijay Diagnostics centre for on time reports in

haematology & histopathology.

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I am thankful to Dr .K.P.Suresh, Scientist (Biostatistics), National Institute of

Animal Nutrition & Physiology, Bangalore, for his kind co-operation in procuring

statistical reports.

Thanks to Mr. D.Vaman Rao, Professor in chemistry & metallurgy for his prudent

discussions on drug analytical results.

My thanks to Central Animal house, Indian Institute Of Science, Bangalore for

providing the experimental animals as needed.

I convey my thanks to Dr.Shivanand, Dr.Shreelatha,Dr.MohanKumar,Vanitha,Vinod,

KVAFSU for their kind assistance in performing experimental study at the locale.

I wish to express my thanks to: Dr.Veerendra Hatti ,Dr.Manjula, Dr.Anuroopa,

Dr.Nischitha,Dr.RamacharyaGudi,Dr.AjithNarayan,Dr.Usharani,Dr.Mamatha,

Dr.Mallamma,Dr.SriMukunda,Dr.C.M.Joshi,Dr.AbdulKareem,Dr.Lajana,Dr.Sunitha,

Dr.Brahmanand,Dr.Rohith,Dr.Srikanth,Dr.ShriRaj,Dr.Manjunath,Dr.PoornimaBhat,

Dr.Prajnami,Dr.SunithaG.S,Dr.SunithaM.L,Dr.Shwetha,Dr.AjayKumar,Dr.Kishor,

Dr.Shrinidhi & Dr.Manjunath for their co-operation during the study period.

My special thanks to my batch mates, friends Dr.Revati.Huddar, Dr.Saritarani

Dr.Sanjeeva gowda Patil, Dr.Naveen.K , Dr.Sandeep.S and Dr.ManjulaC.V for their

adorable assistance through out the study period.

I wish to convey sincere gratitude to my beloved teachers Dr.Ashalatha,

Dr.Ganesh Kumar & Dr.Vijayalakshmi who fed me with blessings.

I am endeared to express my special thanks to Dr.K.V.Guruprasad who stood as

a constant source of inspiration and assisted me throughout the study.

Deep from my heart my gratitude to my lovable parents Smt & Shri G.Rajiv

Shetty who made my dream come true.

I wish to thank my brothers and all my family members for their support in

accomplishing this work.

My sincere thanks to CCRAS for having selected present thesis for grants.

I am sincerely thankful to all teaching staffs, physicians, staff nurses and non-

teaching staffs of T.G.A.M.C. Hospital, Bellary & all those who have helped me directly

or indirectly in completion of this dissertation & for their generous and kind help for

making this work a success.

DR.K.PALLAVI

8

LIST OF ABBREVIATIONS

A.P Ayurveda Prakasha

A.N Adarsha Nighantu

As Arsenic

ATO,As2O3 Arsenic tri oxide

B.P Bhava Prakasha

B.P.N Bhava Prakasha Nighantu

B.R.R.S Bruhad Rasaraja Sundara

d 50 Diffraction 50% for particle Size.

JCPDS Joint Committee For Powder Diffraction

Standards.

K.N Kaiyadeva Nighantu

LD 50 Lethal Dose 50/median lethal dose.

M.N Madanapala Nighantu

MTD Maximum tolerated dose.

NOAEL No observed adverse effect level.

P Phosphorous

R Rasamritam

R.C Rasendra Chudamani

R.N Raja Nighantu

R.T Rasa Tarangini

R.R.S Rasa Ratna Samucchaya

R.J.N Rasa Jala Nidhi

SPF Specific pathogen Free

Y.R Yoga Ratnakara

X-RD X- Ray Diffraction.

XPDF X-ray powder diffraction files

9

TABLE OF CONTENTS Sl No. CONTENTS PAGE

No. I INTRODUCTION. II Aims & Objectives. III Review of Literature. • Malla-A classical Review. • Arsenic & Arsenic trioxide • Karavellaka & its Modern Review • Pharmaceutical Review. • Analytical Review. • Toxicology Review. • Experimental Review. IV Methodology.

Pharmaceutical study. • Malla Shodhana In Dola Yantra Swedana.

Analytical study. • Physico-chemical, AAS, XRD, Particle Size Experimental study.

• Acute Study • Sub-Acute Study

V Results. • Acute Study • Sub-acute Study VI Discussions.

• Acute Study • Sub-acute Study VII Conclusion. VIII Summary. IX Limitations & Scope for the study. X Bibliographic References. XI Annexure.

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LIST OF TABLES SL. NO.

TABLES PAGE NO.

1. Synonyms of Malla according to various authors.

2. Varieties of Malla based on colour.

3. Varieties of Malla based on Appearance.

4. Malla shodhana according to various authors.

5. Gunas of Malla according to various authors.

6. Synonyms of Karavellaka according to various authors.

7. Pharmacological Properties of Karavellaka.

8. Observations during Malla shodhana in Karavellaka Swarasa.

9. Organoleptic Characters of two samples of Malla

10. Summary of Particle Size analysis.

11. Shows Results of X-RD studies.

12. Shows Drugs according to groups in acute study.

13. Shows Calculation of administered dose of each group.

14. Shows Calculation of escalated doses for two groups.

15. Shows the day and number of rats dead in group IV.

16. Shows the day and number of rats dead in group VII.

17. Shows drugs according to groups in Sub-acute study.

18. Shows weight and calculation of doses.

19. Shows the physico-chemical results of Samples

20. Shows Arsenic estimation in the given samples.

21. Shows comparison of body weight between groups.

22. Shows the inter group differences and its significance.

23. Shows the percentage of rats dead in group IV.

24. Shows the percentage of rats dead in group VII.

25. Shows the comparison of Hb between groups.

26. Shows the comparison of TLC between groups.

27. Shows the comparison of DC-P between groups.

28. Shows the comparison of lymphocytes between groups.

29. Shows the comparison of eosinophils between groups.

30. Shows the comparison of monocytes between groups.

31. Shows the comparison of PCV between the groups.

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32. Shows the comparison of RBC between groups.

33. Shows the comparison of Urea between groups.

34. Shows the comparison of Creatinine between groups.

35. Shows the comparison of SGOT between the groups.

36. Shows the comparison SGPT between the groups.

37. Shows the histopathology results.

38. Shows the body weight between groups.

39. Shows the comparison of Hb between groups.

40. Shows the comparison of total Count between groups.

41. Shows the comparison of DC-P between groups.

42. Shows the comparison of DC-L between groups.

43. Shows the comparison of DC-E between groups.

44. Shows the comparison of DC-M between the groups.

45. Shows the comparison of RBC between the groups.

46. Shows the comparison of PCV between the groups.

47. Shows the comparison of SGOT between the groups.

48. Shows the comparison of SGPT between the groups.

49. Shows the comparison of albumin between the groups.

50. Shows the comparison of TPR between the groups.

51. Shows the comparison of creatinine between the groups.

52. Shows the comparison of BUN between the groups.

53. Shows the histopathological results of different groups.

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LIST OF GRAPHS

SL.

NO.

GRAPHS PAGE NO

1. XRD of Raw /Ashuddha Malla

2. XRD of Shuddha Malla.

3. Comparitive XRD peaks between Shuddha and Ashuddha Malla.

4. Diffraction pattern of Particle Size of Shuddha Malla.

5. Diffraction pattern of Particle Size of Ashuddha Malla.

6. Shows the body weight difference in between the groups in acute.

7. Representation of Quantal dose response in group IV.

8. Representation of Quantal dose response in group VII

9. Representation of body weight in between groups Subacute.

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LIST OF ILLUSTRATIONS, FIGURES AND PHOTOGRAPHS

SL.NO FIGURES OR PHOTOGRAPHS PAGE NO 1. Arsenic inhibiting enzymes. 2. Chemical Structure of ATO. 3. Raw Malla. 4. Karavellaka. 5. Ground Karavellaka kalka. 6. Extraction of Karavellaka swarasa. 7. Measured karavellaka swarasa. 8. Malla in pottali. 9. Dola Yantra Swedana. 10. Boiling swarasa after 2 hours. 11. Boiling swarasa after 5 hours. 12. Pottali opened after cooling. 13. Washing Malla pieces in hot water. 14. Powdering Malla. 15. Rats in cage. 16. Cages arranged. 17. Feeding and water for rats. 18. Dosage prepared. 19. Weighing the animals. 20. Analytical balance. 21. Numbering of rats. 22. Tuberculin syringe. 23. Gavaging the dose. 24. Ether chamber. 25. Storage Vials. 26. Preparation of ether chamber. 27. Blood drawing. 28. Capillary tube and blood stored. 29. Centrifuging blood. 30. Semi-auto analyzer. 31. Dissection set. 32. Diethyl ether used for anaesthesia. 33. Micropipettes with serum. 34. Clear Serum in a pipette. 35. Reagent mixing. 36. Rehabilitating rat after anaesthesia. 37. Blood clot after centrifuge 38. Separating Serum 39. Rat in ether chamber. 40. Anaesthetized Rat. 41. Decapitation of rat. 42. Dead rat . 43. Exposing Fascia

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44. Excising the organs

45. Dissection of Brain. 46. Fixing organs in NBF 47. Ready for Histopathology ACUTE STUDY-histopathology. 48. Photo micrographs of Heart tissues. 49. Photomicrographs of Brain. 50. Photomicrographs of Liver. 51. Photomicrographs of Kidney. 52. Photomicrographs of Lungs. 53. Photomicrographs of Spleen. 54. Photomicrographs of Gastric mucosa. SUB-ACUTE STUDY-histopathology. 55. Photo micrographs of Heart tissues. 56. Photomicrographs of Brain. 57. Photomicrographs of Liver. 58. Photomicrographs of Kidney. 59. Photomicrographs of Lungs. 60. Photomicrographs of Spleen. 61. Photomicrographs of Gastric mucosa. 62. Collision in fragmented Particles. 63. Effect of temperature & reaction. 64. Effect of Concentration on collision.

15

ABSTRACT

Title: “Pharmaceutico - Analytical and toxicological study of Shuddha Malla

On Wistar Albino Rats (An experimental Study).”

Background:

Malla is one of the Sadharana Rasa. Shodhana is not only purification but also

pharmaceutical, pharmacological enhancement and detoxification therapy..The drug was

then used to screen the toxicity on Wistar albino rats through Acute and Sub-acute Study.

Objectives: 1) Shodhana of Malla by swedana method.

2) Physico – chemical Analysis of Shuddha Malla.

3) To screen the probable toxicity of Shuddha Malla in repeated doses.

4) To screen the probable toxicity Shuddha Malla in a single dose.

5) To find the median lethal dose of Shuddha Malla & Ashuddha Malla. Methods:

Pharmaceutical Study:

Swedana method for 6 hrs in Karavellaka Swarasa.From this study 47 gm (94%) of

Shuddha Malla was obtained from 50 gm of Raw Malla.

Analytical study:

Physical tests like Ash value etc., Loss on drying, pH, estimation of arsenic,

Particle Size, X-RD studies were conducted on Ashuddha and Shuddha Malla.

Estimation of As% was in significant Purity levels proving to be genuine. An X-

RD study has shown composition of Malla as As2O3 in both the samples with different

standards. Particle size varies which shows an increased size after shodhana

Experimental study:

Toxicity studies both acute and Sub acute was carried out for 14 and 28

days respectively. The Control with the placebo gum acacia was common in both the

study.. The repeated doses of Shuddha and Ashuddha Malla also were administered to

know the significant changes in 2 different respective groups. The escalated doses of both

the drugs were carried out in order to find the LD50 during the acute study. Collection of

blood and necropsy of rats was carried out to extract the data analysis.

Results:

The 10 and 20 times the dose of therapeutic dose of Shuddha Malla proved to be

non-toxic. The repeated daily therapeutic dose of Shuddha Malla also proved to be non-

toxic.

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Conclusion:

◊ Shuddha Malla reveals more of Arsenic %.

◊ The median lethal dose of Shuddha Malla is 16.65mg.

◊ The single dose, repeated therapeutic dose for 14 days and 28 days of Shuddha

Malla is non-toxic.

Key words:

Shodhana, Chemical Analysis, X-RD, Toxicity, escalated dose, median lethal dose.

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INTRODUCTION

Rasashastra dealing with drug pharmacology holds a valid response of drug

designing, formulating, analyzing with a vision to cure diseases. Never medicines have to

be invented, evolved. Probably the result was evolution of mineral medicines after 100s

of years of hard work, observations, experiments and research.

An article JAMA on 15th December 2004, made feel the necessity of safety data

on metallomineral and herbo-mineral formulations used in Ayurveda. Conventionally,

modern toxicology and pharmacology consider all the heavy metals as highly toxic. But

this toxicity the vishathva was tamed by our seers by means of Shodhana. On the contrary

they become potent therapeutically.

Malla is one such wonder Rasadravya used in day to day practice included under

Sadharana Rasa contains Arsenic. On review it was found that pharmaceutical and

analytical studies are carried but not toxicological studies. Hence a positive hypothesis

(H1) is made that, with classical therapeutic dosage the safety of Malla is to be known

and the need of the hour is to prove its safety use through toxicological studies.

So the present work entitled “PHARMACEUTICO – ANALYTICAL AND

TOXICOLOGICAL STUDY OF SHUDDHA MALLA ON WISTAR ALBI NO

RATS(AN EXPERIMENTAL STUDY) ’’is taken up in the interest of producing

experimental base for safety clinical use of Shuddha Malla .

The present study has been categorized into

1. Introduction:

Explains need of the study and hypothesis behind the study.

2. Objectives: Substantiating the mode of study

3. Review of literature:

Reveals the drug review, pharmaceutical review, analytical review

Experimental review and toxicological review.

4. Methodology:

Is divided into three parts

� Pharmaceutical study:

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Includes detailed documentation of Shodhana of Malla by

Karavellaka swarasa carried out.

� Analytical study:

Includes physico – chemical Analysis, estimation of Arsenic in the

Sample along with particle Size and X-Ray Diffraction Study.

Conducted on Ashodhita Malla and Karavellaka Shodhita Malla.

� Experimental study:

Includes screening of probable toxicity of Shuddha Malla and Ashuddha

Malla on Wistar albino Rats. It is in two sections as Acute and Sub-acute

Study.

5. Results:

Includes results of Pharmaceutical, Analytical study and Statistical reports

of Experimental Toxicological Studies both acute and Sub-acute.

6. Discussion:

Includes observations, findings & results of study along with probable

Explanations and theories.

7. Conclusion:

A view of concluding points.

8. Summary:

The whole study is summarized.

9. Bibliographical references:

List of references from classical, modern texts and websites.

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OBJECTIVES

1) To carry out Shodhana of Malla in Karavellaka Swarasa.

2) To carry out Physico-chemical analysis of Shuddha Malla.

3) To carry out Acute toxicity Study of Shuddha Malla and Ashuddha

Malla on Wistar albino rats.

4) To carry out Sub-acute toxicity Study of Shuddha Malla and

Ashuddha Malla Wistar albino rats.

5) To carry out dose-dependent study and evaluate LD 50 of both

Shuddha Malla and Ashuddha Malla.

6) To carry out the relevant Serum and haematological laboratory

investigations.

7) To carry out histopathological analysis to understand organ toxicity.

8) To prove the Safety dose of the drug.

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MALLA

Malla is one of essential Sadharana Rasas1. Most of Rasagranthas considered it

under Sadharana Rasa. Malla as a drug molecule was found in Samhitha kala itself. It

was named as Phenashma and explained along with Haratala and both of them are

considered as Dhatuvisha2.

Nirukti: 3, 4

“Mallati dharati balamiti”

The one which does baladharana, Is strong, robust, to hold, Possess, Excellent.

Nishpatti: 5

The word Malla is derived from ‘Mall’ Dhatu and ‘Ach’ Pratyaya.

Varga6:

It is placed under Sadharana Rasa Varga.

Historical Review:

• Mentioned first as Phenashma along with Haratala as Dhatuvisha2.

• Most of Rasacharyas explained it under Sadharana Rasavarga.

Occurrence7: It is scarcely available along with the ores of Iron, Copper, and Sulphur

etc. It is also available as a component in Haratala and Manahshila.If occurs in mineral

form it has traces of antimony, nickel, silver, iron, bismuth and gold.

Availability 8: In the mineral form in China, Europe etc.In India Hazaaribagh (Bihar),

Chitral (Kashmir) and nearby areas obtained with ores of Makshika etc.The artificial

forms are from chimneys of industries and the places where Arsenopyrites are burnt.

Characteristics9: Malla is crystalline or amorphous substance, white in colour. Its

powder resembles the flour of wheat but is much heavier. The surface of Malla has a

peculiar shine and sometimes there occurs a yellowish tinge.

Physical Properties9: When heated, Malla becomes soft and emits the odour like garlic.

One crystalline variety of Malla is soluble in water, in very small quantity. The crystals

of Arsenic oxide are Octahedral or Monoclinic, of which the latter is unstable. When

heated with intense heat, it evaporates in the atmosphere, directly (sublimation) giving

out garlic odour.

Grahya Malla10: Sphatikabha Malla is of best quality.

Shankhabha or Shwethabha Malla is of better quality.

Haridrabha Malla is of Good quality.

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Vernacular names11:

Kannada : Shankha pashana. Hindi : Shankhia.

English : White Arsenic. Latin : Arsenicum Album.

Gujarati : Somala, Sankhiyo. Marathi : Somalakhara.

Bengali : Sankhavisa. Arabian : Sanmuluphar.

Synonyms: Various synonyms have been given for Malla in Rasashastra classics based

on its colour, appearance, occurrence and action. Ex: Phenashma means white stone2.

Sphatikabha, it looks like Sphatika mani12.

Table No. 1: Shows the Synonyms of Malla.

SYNONYMS R.J.N13 B.R.R.S14 R.R.S15 R.T16 Y.R17

Aakhupashana - - - + -

Darumocha - - - + -

Darumoosha + - - - -

Darumooshaa - - - + -

Gauripashana + - - + -

Hatachoornaka + + + - -

Malla + - + - +

Mallaka - - - + -

Mushaka + - - - -

Peeta - - - + -

Phenashma - - - + -

Phenashmabhasma - - - + -

Sambala + - - + -

Sankhiya - - + - -

Shankhamusha - - - + -

Shankhavisha + - + + -

Somala + - + + -

Sumbalakhara - - + - -

Talasatwa + - - - -

Ullipashana - - - - +

Vikata + + + - -

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Table No. 2: Shows the types of Malla based on colour.

Reference Shweta Rakta Peeta Krishna

A.P18 + - + -

R.T19 + + - -

Y.R20 + + - -

B.R.R.S21 + - + +

Table No. 3: Shows the types of Malla based on appearance.

Reference.

Sphatik-

-abha.

Shankh-

-abha.

Haridr-

-abha.

Dadim-

-abha.

Shwet-

-abha.

Vikata. Hata-

-Choor-

-naka.

R.R.S22 + + + - - - -

A.P 23 - + - + - - -

Y.R 24 - + - + - - -

R.C 25 - - - - - + +

R.J.N26 - + + - - + +

Based on occurrence8:

Natural/ Khanija/ Parvata sambhava and Artificial/ Krutrima.

All the shwetha varieties are krutrima. Rakta, peeta are parvata sambhava.

Need for Malla Shodhana: As Malla is considered as visha27,

Aakhupashana,Shankhavisha, Dhatuvisha28, and now a days it is prepared and available

in artificial form it should be subjected for Shodhana prior to its internal use as medicine.

Malla shodhana: As per different texts there are different Medias indicated.

Table No. 4: Shows the shodhana of Malla according to various authors.

S.N. REF PROCEDURE DRUGS USED METHOD DURATION

1. R.T 29 Pachana Karavellaka swarasa Dola yantra 6hrs

2. R.T 30 Pachana Meghanada swarasa Dola yantra 1 day

3. R.T 31 Pachana Aja dugdha/Ajarasa Dola yantra 1 day

4. R.T 32 Pachana Tankanajala Dola yantra 3hrs

5. R.T 33 Pachana Godugdha Dola yantra 3hrs

6. Y.R34 Pachana Meghanada swarasa Dola yantra 1 day

7. Y.R35 Pachana Kanji/Tankanajala/ Dola yantra 6hrs

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Ajamamsarasa/

Godugdha.

8. Y.R36 Putapaka Kadalikanda Puta paka 6hrs

9. R37 Pachana Godugdha/

Ajadugdha/

Karavellaka swarasa.

Dola yantra 6hrs

10. R.R.S38 Swedana Karavellaka swarasa Dola yantra --

11. R.C39 Swedana Karavellaka swarasa Dola yantra 4hrs

12. R.C40 Swedana Ghananada Swarasa Dola yantra 4hrs

13. R.C41 Swedana Kanji/Tankanajala/

Ajamamsarasa/

Godugdha.

Dola yantra 2 Ghatika

14. R.C42 Putapaka Kadalikanda Putapaka 2 Ghatika

15. R.C43 Pachana Mahishajala/

Mahisha dugdha

Dola yantra 1Yama/

1Yama

16. R.J.N44 Swedana Karavellaka swarasa Dola yantra 1Yama

Malla Marana: 45,46,47,48

1) Take a wide mouthed vessel and keep 2 Palas Suryakshara in it. Above that,

place 2 Palas of Hastidanta churna in such a way that it should look like a heap (i.e. wide

at base and sharp at the tip). Keep this vessel on oven and heat it. When both the drugs

get mixed up and there is no smoke, fire is extinguished. Take out the mixture, mix it

well and preserve it. Take 2 Tolas of this in a Moosha and place 1 Tola Shuddha Malla

Khanda over it. Again it is covered by 2 Tolas of above mixture. Mukhabandhana is done

by using a Sharava and heat is given in a Lavaka Puta. By this white Malla Bhasma is

procured.

2) Take 3 Tolas Rumimastagi Churna. Take one mud pot and keep half (11/2Tola)

of Rumimastagi Churna in that. Above that keep 1Karsha Shuddha Malla and above that

fill the remaining Rumimastagi Churna. Cover that mud pot by using an iron vessel and

do Sandhibandhana.. Immerse the mud pot in Arka dugdha and heat it for 1Prahara. By

this we will get Malla Bhasma.

24

3) Take white ash of Kantharikashta. Take one mud pot. Fill half of it with ashes

of Kantharikashta. Keep 1Karsha Shuddha Malla above that. Remaining Kantharikashta

ashes are then put over it. Keep that pot on oven and Badarakashtagni is given for 2

Yamas. After self-cooling collect the Malla Bhasma.

4) Take Shuddha Malla in a mud pot. Put 5 Karshas of Ajadugdha above that. It

is covered by a cloth. Place this in a pit and cover it by 1Angula mud. Ignite 10

Vanopalas of agni. Like this the procedure is repeated for 21 times. For each Puta, add 5

Tola Ajadugdha. By this we will get Peeta-Aruna coloured Malla Bhasma.

Satwapatana: 49, 50, 51

Take equal parts of Shuddha Malla and Saurashtri churna. Both are kept

in a mud pot. Add 20 Tolas Rambhatoya to it. Place one more mud pot on it and do

sandhibandhana. Keep it on oven and heat it for one Prahara. After self-cooling collect

the material deposited on the upper pot. It is indicated in Shwasa, Kasa, and Jwara. It can

be given along with Ghrita and Sita, Sheeta jala, Dadhi, Dadhyanna.

Malla satwa is prepared as that of Haratala satwa. Malla satwa is Shubhra

varnatmaka.

Essence of Malla is similar to that of Haratala. The essence is pure White,

soothing and destroyer of the three doshas. It is used in the solidification of Mercury and

increases the potency of the same.

Pharmaco-therapeutic properties:

Table No. 5: Shows Gunas of Malla according to various Rasa classics.

SL.NO GUNA R.R.S 52 R.T 53 D.G.V 54

1 Snigdha + + -

2 Laghu - - +

3 Rooksha - - +

4 Teekshna - - +

Rogaghnatha: 55

Destroys kapha vata predominant diseases, and also useful in scorpion bite,

Shwasa, Kasa, Kushta, Shleepadottha Jwara, Yakshma, Sandhivata, Phiranga,

Agnimandya, Vishamajwara, Jeerna Pandu, Pratamaka Shwasa, Hrudshoola,

Hruddaurbalya, Atisara etc. Externally it acts as Kshara. It is Shotha-Santapa-Shaithilya

25

nashaka.When Malla applied to the region of scorpian bite along with water; it kills the

pain in ½ Ghati 56.

Shuddha Malla is Balya, Vrushya, Rasayana, Shwasa, Sheeta Jwara, and

Pandunashaka. It is also useful in Pleehavruddhi, Phiranga, Shleepada, Kushta and

Sandhivata57.It is useful in Vata-Kapha-Sheeta Vyadhis58.

Preparation of Gauripashana (Malla): 59

Haratala rubbed with Castor oil (Eranda Taila) and Lime juice (Nimbu rasa)

and heated by means of a Valuka Yantra, exhibits Red Gauripashana as its

essence.

Matra: 1/120th –1/30th of Ratti.60

1/10th – 1/3rdSarshapa.61

Matra Nirmana Vidhi: 62

Take 1 Gunja – Shuddha Malla.

15 Masha – Maricha Churna.

Q.S. – Shrungavera Swarasa.

Take Shuddha Malla and Maricha Churna in a Khalwa and triturate with

Aardraka Swarasa for 3 days. Prepare 1 Ratti pramana Vatis. 1 vati in the morning and

one in night is given for dosha shamana. In this quantity itself it is added to other

formulations and is used.

Care during Matra Nirmana 63:

1 Ratti Malla causes death. So according to Desha, kala, rogi bala, dosha

etc matra is decided and given. Fatal Dose: 1 Ratti (125mg).

Aamayika Prayoga: 64

Along with various Anupana’s Malla is indicated in various diseases.

� Vasa Satwa/ Vyaghri churna � Purana Shwasa.

� Panchatikta Satwa/churna � Kushta.

� Guduchi churna/Arka churna � Shleepada Jwara.

� Karanjabeeja churna � Cures Vishama Jwara in one day.

26

� Shunthi and Punarnava churna � Amavata and Amavataja Jwara.

� Loha Bhasma with Triphala Kashaya � Purana Pandu.

� Jatipatra and Lavanga churna � Purana and Daruna Phiranga.

� Rasasindhura and Loha Bhasma � Rajayakshma.

� Shankhapushpi churna � Hruddaurbalya.

� Shunthi and Maricha churna � Bhuktamatra samudbhava Atisara.

� Malla triturated with Jala- Lepa � Vruschikadamsha Vishanasha.

Pathya: 65 Milk, Sugar, Butter, grams and other nutritious substances should be taken in

large quantities.

Apathya: 66 Salt, chillies, oil, asofoetida and other hot and spicy things should be

avoided.

Vishishta yogas:

♦ Malla Sindhura. ♦Sameerapannaga Rasa.

♦ Suchikabharana Rasa. ♦Malla Vati

♦ Shankhavishodaya Rasa. ♦ Malla Chandrodaya rasa

♦ Kalanala Rasa. ♦ Malla Garbha Pottali.

♦ Chandeshwara Rasa. ♦ Panchasuta

ARSENIC

Arsenic is ordinarily presumed to be a metal, is in fact a metalloid.67

Arsenic68 is found in the free state but it occurs more commonly in combination with

iron as arsenical iron pyrites or mispickel, FeS2, FeAs2 and as arsenical iron or

leucopyrite, FeAs2.Arsenic forms two oxides-arsenic trioxide,As2O3(the anhydride of

arsenious acid,H3AsO4) and arsenic pentoxide,As2O5(the anhydride of arsenic acid,

H3AsO4).The trioxide is the most important compound of arsenic.

Arsenic69 is found in nature at low levels. It’s mostly in compounds with oxygen,

chlorine, and sulfur (inorganic arsenic compounds). Arsenic in plants and animals

combines with carbon and hydrogen (organic arsenic). Organic arsenic is usually less

harmful than inorganic arsenic. Most arsenic compounds have no smell or special taste.

When arsenic enters the environment: It doesn’t evaporate. Most arsenic compounds can

27

dissolve in water. It gets into air when contaminated materials are burned. It settles from

the air to the ground. It doesn’t break down, but can change form. Inorganic arsenic is a

human poison. Organic arsenic is less harmful. Arsenic damages many tissues including

nerves (peripheral polyneuropathy, axonal degeneration), stomach and intestines, and

skin. All arsenicals except arsine act by inhibiting sulfhydryl enzyme systems required

for cell metabolism and the potency of action depends on the valence of the arsenic atom.

Arsine reacts with hemoglobin to form a very strong hemolytic poison.

Lower levels of exposure to inorganic arsenic may cause nausea, vomiting, and

diarrhoea, decreased production of red and white blood cells, abnormal heart rhythm,

blood vessel damage, a “pins and needles” sensation in hands and feet, painful and

profuse diarrhoea, shock, coma, convulsions and death, irritation, inflammation,

ulceration of mucous membranes and skin, kidney damage. Direct skin contact may cause

redness and swelling.

Chronic toxic effects69: Fatigue, loss of energy, G.I. disturbance, nasal septum

perforation, ulceration in folds of skin, increased pigmentation of skin, appearance of

small “corns” or “warts” on the palms, soles, and torso, exfoliative dermatitis, rashes,

muscular paralyses and atrophy, sensory disturbances, visual disturbances and blindness,

degeneration of liver (cirrhosis) and kidneys, garlic odour to breath, non cirrhotic portal

hypertension. Arsenicosis is a chronic disease due to drinking water containing arsenic.

Arsenic is an ultra-trace essential element (nonmetal) 70:

The deficiency signs are impairment of growth, reproduction, heart function.

The specific function is increased arginine leads to urea and ornithine which helps in

metabolism of methyl compounds71. Arsenic affects arginine, membrane phospholipids

and zinc metabolism. It is less toxic than Selenium, an ultra trace element with an

established role72. Selenium in toxic amounts is corrected by Arsenic which has a positive

effect. 73

The order of toxicity of arsenic compounds is74 :Arsines (As (III))>arsenite (As

(III)) >arsenate (As (V)) and arsenic-organic acids (As (V)).Arsenic which is found

mainly in liver,lungs,kidneys and intestinal walls, is readily absorbed if water

soluble.Arsenic is also toxic by replacement of Phosphorous in ATP readily absorbed if

water soluble.

Lethal dose of more toxic forms of arsenic is 125 mg per kg body weight.

28

But for less toxic forms, say arsenic in drinking water, larger quantities are

necessary, i.e, 100-200 mg per kg body weight. An antidote for arsenic is BAL, British

Anti Lewisite.74

The toxic effect of arsenic trioxide is by attacking –SH groups of an enzyme, thereby

inhibiting enzyme action75.

SH

Enzyme + As-O Enzyme As-O+2OH

SH

Fig 1: Showing the As and enzymatic action.

Compounds of Arsenic76: In the early part of the present century Ehlrich began the

investigation of organic arsenic compounds, with the object of preparing substances

which might be of the value in the treatment of spirochetes or protozoan diseases such as

syphilis ,yaws, relapsing fever ,sleeping sickness, and amoebic dysentery. The real

problem was to obtain a substance which would be highly active in exterminating the

parasites, and yet would have as low toxicity as possible to the host. As a result of a great

deal of research, a number of arsenical compounds were selected that had these properties

to a high degree. They proved of great medicinal value, especially in the period before

the introduction of antibiotics.

Haematology: 77

Serum Arsenic level is 7 mg/ dl.

Urinary excretion of Arsenic 100mg/day or more indicates arsenic poisoning.

Potency78

The LD50 for pure arsenic is 763 mg/kg (by ingestion) and 13 mg/kg (by intraperitoneal

injection). For a 70 kg (~155 lb) human, this works out to about 53 grams (less than 2

ounces). However, compounds containing arsenic can be significantly more toxic.

29

ARSENIC TRIOXIDE

Arsenic trioxide which was official in all earlier pharmacopoeias, is the

commonest compound of arsenic, and is obtained as a byproduct in many metallurgical

operations, particularly in the extraction of tin, nickel,and cobalt. At least three forms of

arsenic trioxide are known, and the ordinary substance is a mixture of an amorphous

vitreous form and an octahedral form.79

HISTORY OF ARSENIC TRIOXIDE (AS2O3)80

Because Of its significant medicinal properties, arsenic has been used as

therapeutic agent since 2,400 years .In the 15th century William Withering who

discovered digitalis was a strong proponent of arsenic-based therapies. He argued

“Poisons in small doses are best medicines; and the best medicines in too large doses are

poisonous. Pharmacology texts of 1880’s describe the use of arsenical pastes for cancers

of skin and breast and arsenous acid was used to treat hypertension, bleeding gastric

ulcers, heart burn , chronic rheumatism.Arsenics reputation as a therapeutic agent was

enhanced when Noble laureate Paul Ehlrich developed ‘Salvarsan’ an organic arsenical,

for treating Syphilis and Trypanosomiasis.Arsenics antileukemic activity was reported in

late 1800,s.In 1878 a report from Boston City Hospital described the effect of Fowlers

solution on reduction of white blood cell counts in two normal people and one patient

with leucocythemia.Subsequently Arsenic trioxide was administered as a primary

antileukemic agent until it was replaced by radiation therapy. In one report from China

the monotherapy of Arsenic trioxide produced a complete clinical response in 9 of 10

with relapsed APL.Consequently, TRISENOX (As2 O3) was approved for the relapsed or

refractory APL by US FDA in September 2000.

Arsenic trioxide81 was the active ingredient of the liquor arsenicals or arsenical solution

of 1932, 1948 and 1953 pharmacopoeias. The preparations were widely prescribed in

tonics.

Other names :-

� Arsenic(III) oxide

� Arsenolite

� Arsenic sesquioxide

� Arsenicum album

� Arseneous oxide

� Arseneous anhydride

30

Properties: Arsenic trioxide is a white or transparent solid in the form of glassy,

shapeless lumps or a crystalline powder that resembles sugar. It has no odor or taste. It

forms readily when elemental metallic arsenic is heated to high temperatures or burned.

Physical and Chemical properties82

� Molecular formula

As4O6,As2O3

� Molar mass

197.841 g/mol Fig 2: Arsenic trioxide

� Appearance : White solid Opaque mass.

� Odor : No Odor.

� Taste : No taste/ slightly metallic taste.

� Hardness : 3 – 4

� Specific gravity : 5.6 – 5.8

� Molecular weight : 197.84 Daltons

� Boiling point (760 mm Hg) : 869ºF (465ºC)

� Sublimes at : 379ºF (193ºC)

� Melting point : 594ºF (312ºC)

� Vapor pressure : 66.1 mm Hg at 594ºF (312ºC)

� Density and phase : 3.86 g/cm³, solid.

� Solubility in water : 2 g/100 ml (25°C)

� Acidity (p''K''a) : 9.2

� Mohs hardness : 1.5

� Refractive Index : 1.755

� Lattice constant : 11.074�Å

� Crystal structure : Cubic (α) <180°C

Monoclinic (β) >180°C

� Dipole moment : Zero

� Specification : As2O3-99.5%,Fe-0.05%,moisture-0.5%

� Standard enthalpy

31

of formation ∆f''H''osolid : −657.4 kJ/mol

� Water solubility : Low solubility in water (37 g/L at 20ºC, 115 g/L at

100ºC); slightly soluble in alcohol; soluble in dilute HCl solutions.

� Flammability : not flammable, but emits highly toxic arsine gas and

oxides of arsenic fumes when burned.

Arsenious oxide on sublimation forms a transparent glassy mass which gradually

becomes opaque in presence of moisture. It is a colorless, odorless, taste less substance83.

Arsenic trioxide is an amphoteric oxide which shows a marked preponderance for

its acidic properties. It dissolves readily in alkaline solutions to give arsenites. It is much

less soluble in acids, but will dissolve in hydrochloric acid to give arsenic trichloride or

related species. It reacts with oxidizing agents such as ozone, hydrogen peroxide and

nitric acid to give arsenic pentoxide, As2O5: the reaction with hydrogen peroxide can be

explosive. It is also readily reduced to arsenic, and arsine (AsH3) may also be formed.

Related Compounds:

Other anions: Arsenic Trisulfide

Other Cations: Phosphorous trioxide, Antimony trioxide.

Varieties84:

� Vitreous amorphous form: Specific gravity 3.74 which is more or less

transparent and melts without volatization at

approximately 2000C.

� Crystalline octahedral state: Melting point 2750C and specific gravity

3.69. It sublimates without fusion.

� Monoclinic variety : Melting point 3150C and specific gravity 3.85.

In industries AS2O3 is purified by repeated sublimations in iron pots. A

precaution is taken that the AS2O3 is not reduced to As, which would then eat up the iron

pots and more over it would fall into furnace with giving poisonous gases85.

Tests86

The following are the main tests of arsenic.

� Arsenic compounds when heated on charcoal give a white encrustation far

from the assay and at the same time fumes having a garlic odor are emitted.

32

� Heating in the open tube arsenic compounds gives a white sublimate which is

volatile on heating

� Heated in the closed tube some arsenic compounds give shiny black sublimate

the arsenic mirror; most arsenates give a similar mirror when heated with

charcoal or sodium carbonate in the closed tube.

Pharmacological actions87:

Organic arsenicals used in chemotherapy of trypanosomiasis, amoebiasis and

trichomoniasis in the form of carbarsone, tryparsomide, cycobiarsol, melarsoprol. They

are no longer in the treatment of syphilis.

Pharmacology: 88

1. Inorganic arsenicals used mainly as rodenticides, herbicides and insecticides.

2. Organic arsenicals in the chemotherapy of Trypanosomiasis. They were in the

mainstay of treatment of syphilis.

Absorption, fate and excretion: 89

The inorganic forms of arsenic are more toxic than the organic forms and the

trivalent forms are more toxic then the pentavalent forms. Arsenic can be inhaled,

absorbed through the skin, or absorbed in the GI tract after ingestion. After a very small

dose of arsenic (like those experienced daily by most people) most of the absorbed

inorganic arsenic undergoes methylation, mainly in the liver, to monomethylarsonic acid

and dimethylarsinic acid which are excreted, along with residual inorganic arsenic in the

urine. However, if the dose of arsenic is very large, the elimination half-life is

prolonged.Once absorbed, arsenic rapidly combines with the globin portion of

haemoglobin and therefore localises in the blood. There is minimal penetration of the

blood-brain barrier, and within 24 hours arsenic redistributes itself to the liver, kidney,

spleen, lung and GI tract, with lesser accumulation in muscle and nervous tissue

Toxbase summary 90

Toxicity: Generally less acutely toxic than soluble arsenic salts. A patient has

died after ingesting 2 g.

Death from acute arsenic poisoning is usually caused by irreversible

circulatory insufficiency, but if the dose is not large enough to kill the patient a number

33

of secondary effects can be seen 2-4 weeks after ingestion of the poison. These include

hair loss, Mee’s lines (white transverse lines seen on the nail plate up to a year after

arsenic intoxication), sensorimotor peripheral neuropathy (may develop within a few

hours of ingestion but usually seen 2-8 weeks after exposure), skin changes (as in chronic

poisoning) and possible chronic renal failure.

Fatal dose91: 200 – 300 mg.

Fatal period91: 12-48 hrs. The shortest period is 45mins.

Prehospital Management92

Quickly assess for a patent airway, and ensure adequate respiration and pulse.

Maintain adequate circulation.

Skin Exposure: Wash exposed skin and hair with mild soap and water, and rinse

thoroughly with water. Use caution to avoid hypothermia, particularly with children and

the elderly.

Eye Exposure: Flush exposed or irritated eyes with plain water or saline for at least 15

minutes. Remove contact lenses if easily removable without additional trauma to the eye

Ingestion: Do not induce emesis. The effectiveness of activated charcoal is questionable,

but administration of activated charcoal as an aqueous slurry in persons who are awake

and able to protect their airway is recommended pending further evaluation in cases of

ingestion of unknown quantities. Activated charcoal is most effective when administered

within 1 hour of ingestion. At 1 gram per kilogram (gm/kg), the usual adult dose is 60-90

grams (g), and the child dose is 25-50 g. A soda can and straw may be of assistance when

offering charcoal to a child. Complications include emesis and aspiration.

Persons with evidence of significant exposure and all persons who have ingested

arsenic trioxide should be transported to a medical facility for evaluation.

Hospital/Emergency Room Management

34

Evaluate and support the airway, breathing, and circulation as appropriate.

Establish intravenous access in symptomatic patients and monitor cardiac rhythm.

Antidote93: Hemodynamic stabilization and gut decontamination are key factors in the

initial management of acute arsenic intoxication. Chelating agents administered within

hours of arsenic absorption may successfully prevent the full effects of arsenic toxicity.

1. Dimercapol /BAL

2. Freshly prepared gelatinous hydrated ferric oxide is given which acts with

arsenious trioxide to form ferric arsenite and it is a harmless salt.

3. DMSA (succiner) – Meso 2,3 Dimercapto succinic acid

4. DMPS (Unithiol) – 2, 3 Dimercapto propane – 1 Sulfonate.

Both 3 & 4 decreases the arsenic content in the tissue.

Arsenic trioxide uses94:

• Starting point for the manufacture of arsenic-based pesticides. (Sodium arsenite,

sodium arsenate, sodium cacodylate).

• Starting point for the manufacture of certain arsenic-based pharmaceuticals

(Neosalvarsan) and veterinary products.

• Decolorizing agent for glasses and enamels and Preservative for wood.

• Hydrogen recombination poison for metallurgical studies.

• Starting point for the preparation of elemental arsenic, arsenic alloys and arsenide

semiconductors.

• Use as a cytostatic in the treatment of refractory promyelocytic (M3) subtype of

acute myeloid leukemia. The drug is available as Trisenox® ampules; each

containing 10mg to be diluted for i.v. infusion.

• Medical applications: Arsenic trioxide under the trade name Trisenox

(manufacturer: Cephalon) is a chemotherapeutic agent of idiopathic function used

to treat leukemia that is unresponsive to first line agents. It is suspected that

arsenic trisulfide induces cancer cells to undergo apoptosis. Due to the toxic

nature of arsenic, this drug carries significant risks.

• The enzyme thioredoxin reductase has recently been identified as a target for

arsenic trioxide.

35

KARAVELLAKA

INTRODUCTION

The drug Karavellaka (Momordica Charantia) belongs to Shaaka Varga,

Cucurbitaceae family is seen throughout in India and also in Malaya, China, tropical

Africa and America, up to an altitude of 1500m.

. It is a Climber bearing yellow coloured flowers. It is cultivated in gardens

everywhere in India for its fruits. It is used as vegetable. Fruit is bitter in taste. Fruits

bear numerous triangular tubercles like Crocodile’s back. All parts of this plant can be

used.

HISTORY:

Karavellaka is mentioned in all Brahattrayis and almost all Nighantus. It is used

by Unani Hakim’s also.

SAMHITA PERIOD:

� Karavellaka is mentioned under Tikta Skandha95.

� Karavellaka is indicated for Vrana.96 and can be used to improve vision.96 .

� Inclusion of Karavellaka in Aragvadhadi Gana97 .

� Karavellaka is also included in Tikta Varga97.

� Karavellaka is one of shaakhas which is having bitter taste and mitigates Kapha

and pitta98

NIGHANTU PERIOD 99,100,101,102,103

We find the reference of Karavellaka and its qualities in :

1) Adarsha Nighantu 4) Kaiyadeva Nighantu

2) Bhavaprakasha Nighantu 5) Raja Nighantu

3) Madanapala Nighantu

NIRUKTI 104

“Kaarena prayatnena yashtyadikamavalambya vellati chalati iti Karavellaha

Vellam chalane│

Karam jwaradinadyam Prativellati iti”

It motivates all its sources/destroys diseases like Jwara.

NOMENCLATURE:

Sanskrit name � Karavellaka

Latin name � Momordica Charantia

36

Kingdom � Plant Kingdom

Class � Dicotyledons

Sub-Class � Polypetalae

Series � Calyciflorae

Order � Passiflorales

Family � Cucurbitaceae

Genus � Momordica

Species � Charantia

Kula � Koshataki Kula

Gana:

Sushrutokta Gana � Aragvadhadi Gana

Varga:

Sushrutha Samhitha � Tikta Varga

Charaka Samhitha � Shakha Varga

Astanga Hridaya � Shakha Varga

SYNONYMS

Table No: 6 Shows Synonyms of Karavellaka.

SYNONYMS A.N105 M.N106 B.N107 R.N108 K.N109

Brihatvalli - + - - -

Kandakatuka - - - + +

Kandeera - - - + +

Karavella - + + - -

Karavellaka + - - - -

Karavelli - + + + -

Kathila - - + - +

Katila + + - - -

Noyavalli - - - + -

Patu - - - + -

37

Sukanda - - - + +

Sushavee + - - - -

Ugrakanda - + - + +

Varivalli - + - - -

VERNACULAR NAMES

Sanskrit � Karavellaka, Pitapushpa, Sushavi, Toyavalli,

Ambuvallika, Chiripatra, Brihadvalli, Padu,

Karavalli, Sukandaka, Katilla.

Arabic � Qisaulbarri

Assam � Kakiral,Kakral

Bengali � Barmasiya, Jethuya, Karala, uchete

English � Carrilla fruit, bitter gourd

Gujarati � Karela, Karelo, Karelu

Hindi � Karela, kareli, Karol

Kannada � Hagalakayi

Konkani � Karatim

Malayalam � Kaippa, Kaipavalli, Pavakka Cheti

Marathi � Karalu,Karli

Orissa � Kalara,Salara

Punjabi � Karela, Karila

Simhalese � Karawila, Battukarawilla

Tamil � Pavakkay, Paval, pakar

Telugu � Kakara

Tulu � Kanchala

Urdu � Karella

SCIENTIFIC CLASSIFICATION 110:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Cucurbitales

Family: Cucurbitaceae

38

Genus: Momordica

Species: M. charantia

Synonyms110: Momordica chinensis, M. elegans, M. indica, M. operculata, M. sinensis,

Sicyos fauriei.

Description:- 111

Momordica charantia is a tropical and subtropical vine of the family

Cucurbitaceae, widely grown for edible fruit, which is among the most bitter of all

vegetables. English names for the plant and its fruit include bitter melon or bitter gourd.

The herbaceous, tendril-bearing vine grows to 5 m. It bears simple, alternate

leaves 4-12 cm across, with 3-7 deeply separated lobes. Each plant bears separate yellow

male and female flowers.The fruit has a distinct warty looking exterior and an oblong

shape. It is hollow in cross-section, with a relatively thin layer of flesh surrounding a

central seed cavity filled with large flat seeds and pith. Seeds and pith appear white in

unripe fruits, ripening to red; they are not intensely bitter and can be removed before

cooking. However, the pith will become sweet when the fruit is fully ripe, and the pith's

color will turn red. The pith can be eaten uncooked in this state, but the flesh of the melon

will be far too tough to be eaten anymore. The flesh is crunchy and watery in texture,

similar to cucumber, chayote or green bell pepper. The skin is tender and edible. The fruit

is most often eaten green. Although it can also be eaten when it has started to ripen and

turn yellowish, it becomes more bitter as it ripens..Bitter melon comes in a variety of

shapes and sizes.

Main Actions 111 Other Actions111 Standard Dosage111

� Kills bacteria � reduces inflammation

� Kills viruses’ � fights free radicals Decoction: 1 cup 1-2 times daily

� Kills cancer cells � enhances libido Tincture: 1-3 ml twice daily

� Kills leukemia cells � cleanses blood Capsules: 1 g twice daily

� prevents tumors � detoxifies

� Treats diabetes � expels worms

� reduces blood sugar � balances hormones

� reduces blood pressure � enhances immunity

� lowers body temperature � mildly laxative

� Lowers cholesterol � promotes milk flow

39

Pharmacological activities 112:

Antispasmodic, antioxytocic ,hypoglycemic ,mild cholinergic blocking

activity,abortificent,antipyretic,mild-hypotensive,anti-bacterial,uterine

stimulator,anthelminthic,insecticidal,anti-viral,anti- lipolytic

antidiabetic,antimalarial,antispermatogenic,antisteroidogenic,androgenic,anti-

ulcerogenic,antifungal,juvenomimetic.

PROPERTIES :

Table no 7: shows the Pharmacological Properties of Karavellaka.

RASA GUNA VEERYA VIPAKA

Kaiyadeva Nighantu113 Tikta, Katu - - Katu

DravyaGuna114 Tikta, Katu Laghu, Rooksha Ushna Katu

Raja Nighantu115 Katu, Tikta - Ushna -

Astanga Hridaya116 Katu,Tikta - Ushna Katu

Adarsha Nighantu117 Tikta - Sheeta Katu

DOSHAGHNA KARMA

The drug is said to be,

Pittashamaka, Kapha Pittashamaka, Tridoshashamaka

ROGAGHNA KARMA

♦ Madhumeha nashaka ♦ Shothahara

♦ Jwaraghna ♦ Vishaghna

♦ Kushtahara ♦ Vranaghna

♦ Krimighna ♦ Netra roga

♦ Arsha ♦ Karna shoola

♦ Pittaja mastishka shoola ♦ Visuchika

♦ Nadivrana ♦ Visarpa

♦ Pandu ♦ Vatarakta

♦ Kamala ♦ Shwasa

♦ Kasa ♦ Shleepada

♦ Galaganda ♦ AmlaPitta

40

AUSHADHA MATHRA

The dose of the Karavellaka according to its different forms is as follows :-

Swarasa - 10-30 ml

Choorna - 3-6 masha

Including emesis- 100ml

For Madhumeha - 3-6gms of Choorna of fruit with water

For Ashmari - 3 tola of leaf Juice.

AMAYIKA PRAYOGA

EXTERNALLY

• The whole plant is applied in scabies and other cutaneous diseases, in

combination with the oil of Cinnamon etc.

• In Leprosy, fruit is used.

• In Night blindness - juice of leaves applied around the orbit with black pepper.

• In burning of soles - juice of leaves is rubbed to soles 2 or 3 times a day.

INTERNALLY

• It acts as stomachic tonic, anthelmintic, Rheumatism, gout, fruit is used.

• Anthelmintic - fruits and leaves are used.

• Emetic, purgative - juice of leaves with purgatives.

• It is also used in Jaundice, as an appetizer, blood purifier, dysmenorrhea, urinary

disorder, asthma, cough, obesity etc.

• In diabetes-juice of raw fruit, ½ ounces at a time, once daily in empty stomach

early in morning.

PHYTO CHEMISTRY 118: Bitter melon contains an array of biologically active

plant chemicals including triterpenes, proteins, and steroids. One chemical has clinically

demonstrated the ability to inhibit the enzyme guanylate cyclase that is thought to be

linked to the cause of psoriasis and also necessary for the growth of leukemia and cancer

cells. In addition, a protein found in bitter melon, momordin, has clinically demonstrated

anticancerous activity against Hodgkin's lymphoma in animals. Other proteins in the

plant, alpha- and beta-momorcharin and cucurbitacin B have been tested for possible

anticancerous effects. Two of these proteins-alpha- and beta-momorcharin-have also been

reported to inhibit HIV virus in test tube studies. In one study, HIV-infected cells treated

41

with alpha- and beta-momorcharin showed a nearly complete loss of viral antigen while

healthy cells were largely unaffected. The chemicals that lower blood sugar include a

mixture of steroidal saponins known as charantins, insulin-like peptides, and alkaloids.

Its Constituents 118: Alkaloids, charantin, charine, cryptoxanthin, cucurbitins,

cucurbitacins, cucurbitanes, cycloartenols, diosgenin, elaeostearic acids, erythrodiol,

galacturonic acids, gentisic acid, goyaglycosides, goyasaponins, guanylate cyclase

inhibitors, gypsogenin, hydroxytryptamines, karounidiols, lanosterol, lauric acid, linoleic

acid, linolenic acid, momorcharasides, momorcharins, momordenol, momordicilin,

momordicins, momordicinin, momordicosides, momordin, multiflorenol, myristic acid,

nerolidol, oleanolic acid, oleic acid, oxalic acid, pentadecans, peptides, petroselinic acid,

polypeptides, proteins, ribosome-inactivating proteins, rosmarinic acid, rubixanthin,

spinasterol, steroidal glycosides, stigmasta-diols, stigmasterol, taraxerol, trehalose,

trypsin inhibitors, uracil, vacine, v-insulin, verbascoside, vicine, zeatin, zeatin riboside,

zeaxanthin, and zeinoxanthin are all found in bitter melon.

Physical Constituents in the Fruit: 119

Foreign matter-Nil

Total Ash-Not more than 8.5%

Acid-insoluble Ash-Not more than 0.6%

Alcohol soluble extractive- Not less than 6%

Water –soluble extractive-Not les than 28%

Dosage and administration 118 :

Bitter melon may be consumed in the following ways: as food, a decoction of as

much as 3 1/3 ounces (100 ml), or 2 ounces (60 ml) of fresh juice (daily). Though still

bitter, tinctures of bitter melon (1 teaspoon [5 ml] two to three times per day) may also be

used. The above dosages would be appropriate for diabetics.

Side effects and interactions 118:

Excessive ingestion of bitter melon juice (several times more than the amount

recommended above) may lead to diarrhoea and abdominal pain. Consuming excessive

amounts of the seeds may be linked with fever, headache, and coma. Pregnant women

should not use bitter melon. Hypoglycemia, or low blood sugar, may be negatively

affected by bitter melon because it may trigger or worsen the condition.

42

VISHISHTA YOGAS:-

♦ Sheeta nagankuram ♦ Jwarari rasa

♦ Kacchuradi taila ♦ Brihath Vishama jwarantaka lauha

♦ Maha vishagarbha taila ♦ Vidya vallabha rasa.

43

PHARMACEUTICAL REVIEW

‘Kushala Rasa Karmani’ is the quality of a RasaShastraghna120.The

integral Part of Rasashastra lays in the successful pharmaceutical process .Among distinct

Samskaras like Shodhana, Marana, and Jarana etc. Shodhana stands par excellence.

So here is a gross view on the Swedana process of the drug. . In a broader sense the

above said basic pharmaceutical procedure can be denoted under the heading of the

procedures adopted and the Yantras used in the study. Practically the above said

procedure will be carried out and hence to aid the theoretical clarity a brief note has been

drawn vide infra.

PROCEDURE ADOPTED:

Samskara121: ‘Gunantharaadhaanam’ i.e enhancement of the qualities of a drug

and ‘Doshaapanayanam’ i.e to remove the unwanted impurities. One has to apply

Various Techniques such as122

• Toya Sannikarsha- to the vicinity of water.

• Agni Sannikarsha- to the vicinity of fire.

• Toyagni sannikarsha-to the vicinity of both fire and water.

• Shoucha- to wash or cleanse.

• Manthana- to Churn.

• Bhajana-by using the utensils.

• Desa- the place/region of preparation.

• KalaPrakarsha- time period taken.

• Bhavana- by Trituration.

Shodhana123: “Shodhayati iti” (Shudh +nich+pluth)

That which purifies,cleanses and makes Pure.

Need for Shodhana 124: Just like a cloth that cannot be coloured until it is washed or

cleansed.In the same way the process of purification is a must in case of raw drugs.

Shodhana 125,126,127 is the pharmaceutical procedure in which all the drugs of

origin from metals , minerals, animals and herbs are subjected, before subjecting them to

Marana or before administration in case of some Rasa Dravyas like Malla, Shilajatu,

Gairika, Kasisa etc.

44

The literal meaning of shodhana is purification. But in Rasashastra, shodhana is

not merely purification, but is a samskara, which essentially brings out modifications or

alterations in properties along with purification.

Historical Background:

Bruhathrayis do not mention about the special methods of shodhana of metals

and minerals. It is only in the golden era of Rasashastra and Nighantu period dealt

elaborate description of shodhana of minerals and metals as well as herbal drugs

specifically.

Most of the raw materials in Rasashastra are native from earth. So the very chance

of impurities, toxicity, heterogeneous qualities, mixing of other substances and unwanted

qualities to a large extent. Now a days some of the Rasadravyas are artificially prepared.

So shodhana is indicated to eliminate all such toxic qualities, induce and enhane certain

special qualities which are essential for the easy assimilation of the material in the living

body.

Definition: 128,129,130

“Shodhanam karma vijneyam dravya dosha nivaaranam”

The process which eliminates the blemishes is called shodhana.

“Uddhishtairoushadhaihi saardham kriyate peshanaadikam

Mala vichchittaye yattu shodhanam thadihochyate”

When a substance is subjected to trituration etc with required medicine

for removal of unwanted materials or impurities is known as shodhana.

“Loha dhatu rasaadeenamudithaihi aushadhaihi saha

Swedanam mardanam chaiva tailado daalanam tatha

Doshaapanuttaye vaidyaha kriyate shodhanam hi tat”

Subjecting the Loha, Dhatu, Rasoparasas etc to the procedures like swedana,

mardana etc with the prescribed medicines, Dhalana in tailadi dravadravyas to remove

the Doshas is termed as shodhana.

The meaning of shodhana can be:

♦ To Clean ♦ To Dehydrate

45

♦ To Distil ♦ To Polish

♦ To Peel ♦ To Dehusk

♦ To Clarify ♦ To Filter

♦ To Wash ♦ To Purify

Objectives of Shodhana:

• To make metallic substances suitable for Marana.

• To remove Physical and Chemical impurities & Regulation of Physico-

chemical attributes.

• Separation of admixtures & Elimination of harmful matters from the drug.

• Metals are made free from blemishes.

• Reduce or minimize toxic effect.

• Make metal or mineral soft and brittle.

• Reduction in particle size.

• Make substance suitable for further processing.

• Transformation of attributes or imbue organic qualities to inorganic

substances.

• Increasing the potency of the drugs.

• Modification of undesirable physical properties of the drug.

• Conversion of some of the characteristics of the drug to different stages.

• Enhancement of therapeutic action.

• Conversion of drugs from heterogeneous state to homogenous state.

• Corrects the imperfections.

Types of shodhana:

1. Samanya shodhana

2. Vishesha shodhana

Samanya shodhana: The common method used to purify a group of drugs is known as

Samanya shodhana. This process eliminates general impurities of metals and minerals

and converts them into powder which is essential for further process.

Ex: Samanya shodhana of dhatus by repeatedly quenching various liquid media.

Vishesha shodhana:It is done specifically for a particular drug with the view of

purifying it with the help of particular or specific shodhana material as well as procedure.

46

Ex: Swedana of Haratala in Kushmanda Swarasa by Dolayantra method.

Various methods of Shodhana

Various methods of shodhana are mentioned in Rasa classics.

♦ Swedana ♦ Prakshalana

♦ Mardana ♦ Bhavana

♦ Murchana ♦ Shoshana

♦ Patana ♦ Bharjana

♦ Avapa ♦ Samyoga

♦ Nirvapa ♦ Vibhaga

♦ Dalana ♦ Nimajjana

♦ Galana ♦ Pachana

♦ Dhavana.

♦ Washing ♦ Desumption ♦ Shifting

♦ Decoloration ♦ Bleaching ♦ Elutriation

♦ Boiling ♦ Maceration ♦ Pulverization

♦ Lixiviation ♦ Dipping ♦ Percolation

♦ Sublimation ♦ Dialysis ♦ Chemical combination

These are also some of the methods of shodhana for many Rasadravyas. The

dravyas used might be kshara,amla,sneha etc.

SHODHANA YANTRA:

Different types of Yantras are mentioned in the Rasa classics for different

shodhana procedures. Viz Dolayantra,Damaru Yantra, Khalva Yantra, Urdhwapatana

yantra, Sharava,Sthalika,Sandamsani, Darvi etc.Shodhana depends upon: Structure,

Composition, Impurities, Qualities, Action of the drug.

In the present study, for the shodhana of Malla, the Swedana method of shodhana has

been adopted as per classical text.

Swedana:-

Definition132: “Gharmodhgama roopa upakramam”

“Samskaram mala shithila aapaadanaarthanam”

The word ‘Swedana’ literally means causing to perspire, the act of sweating or

perspiring. Boiling the drug by suspending in liquids (bathing) i.e. acids or alkalis or any

medicines, decoctions with the help of Dolayantra (a special apparatus for boiling) is

known as Swedana.133

47

“Kshaaramlairoushadhairvaapi dola yantre sthitasya hi

Pachanam swedanaakhyam syanmalashaithilyakarakam”

Uses:

• Impurities soluble in acid or alkali are eliminated.

• Mala shaithilyata.

Different Yantras have been used for shodhana procedures and associated works

in the present study. They are 134

1. Dolayantra 2.Khalva yantra.

Dolayantra 135,136,137:

It is called Dola yantra because here the drug bundle to be subjected to

Swedana is hung or suspended in a vessel containing liquid like a Dola (swing).A pottali

is prepared using three folds of cloth in which the drug is placed.Take a Pot and fill it

with the drava dravya prescribed to half of the pot . A strong rod should be placed

transversely after making small holes on either sides of the pot near its mouth. On this

rod, the pottali should be hanged with the help of a string till it is submerged in the liquid

at the bottom of the jar. Ignite by placing the yantra over fire. Thereafter fomentation

initiates.

Applied aspect:

It is used to apply heat treatment to the drugs either through the boiling liquids or

through the vapours for a specified time to loosen its impurities. The advantage of

applying swedana by this method is to put the drug directly in contact with the boiling

liquid which may help the drugs to be softened first and then allowing their soluble

impurities to be dissolved into the boiling liquids used for and thus making the drugs free

from some of the impurities135.

According to media used for Swedana, acidic, alkaline, volatile impurities get

eliminated.

Khalva Yantra :

� “ yantram dronyaakaaram peshanopayogi yantram”138

It is a hollow, round or boat shaped apparatus made of iron, stone, glass or

porcelain as per need. For mercurial operations, Khalvas made out of iron are

preferred while for preparing pistis, bhasmas and formulations, shodhana of

certain Rasadravyas Khalvas made out of stone are preferred.

Generally Khalvas are of two types i.e. Vartula and Dronyakriti.

48

• Vartula Khalva is made of porcelain or stone. It should be 12 angula

in radius, 4 angula in depth and 8 angula in length.

• Dronyakriti or boat shaped Khalvas are generally used for mercury

processing and made of iron or stone. Their height varies from 9” to

16”, length 16” to 24”, breadth 9” to 10”, depth 6” to 7” and thickness

of their edges is 2” 139

Uses: It is used for grinding, rubbing, triturating or mixing of drugs and liquids139.

In the present study mortar and pestle made of porcelain were used for making small

pieces of Malla before subjecting to shodhana and after shodhana to make it a soft

powder.

ANALYTICAL REVIEW

To evaluate the quality of the drugs, the scientific validation plays an important

role. As a part of standardization, the classically processed Rasadravyas, prepared

Rasayogas will be subjected to mandatory baseline and current instrumental analysis. In

the present study the two samples of Malla i.e. Ashuddha Malla and Karavellaka

Shodhita Shuddha Malla are screened for physicochemical and instrumental

standardization methods. So the review of analytical procedures is carried out vide infra.

Physico – Chemical tests:

Ash value, Acid insoluble ash,Water insoluble ash, Loss on drying,Loss on

ignition, and pH value are some of the physicochemical tests, employed in the present

study. The brief review of the same is made herewith as under.

1) Determination of Ash value140, 141:

Definition of Ash: The residue remaining of incineration is the ash content of the drug,

which represents the inorganic salts naturally occurring in drug or adhering to it or

deliberately added to it as a form of adulteration.

Method:

Total ash is designed to measure the total amount of material produced after

complete incineration of the ground drug at as low temperature as possible (about 4500)

to remove all the carbons. 2 to 3gms of the air dried crude drug has to be accurately

weighed in the tarred Platinum or Silica dish and incinerate at a temperature not

exceeding 4500 C until free from carbon. Cool and weigh. If a carbon free ash cannot be

obtained exhausts the charged mass with hot water, residue to be collected on ash less

49

filter paper, incinerate the residue and filter paper until the ash is white or nearly so.

Percentage of ash to be calculated with reference to the air-dried drug.

Applied aspect:

Controlled incineration of crude, results in an ash residue consisting of inorganic

material. Total ash value represents the inorganic salts naturally occurring in drug or

adhering to it or deliberately added to it as a form of adulteration. This value varies

within fairly wide limits and therefore an important parameter for the purpose of

evaluation of crude drugs. The total ash usually consists of carbonates, phosphates,

silicates and silica. High ash value is indicative of contamination, substitution,

adulteration or carelessness in preparing the drug

2) Determination of Acid insoluble ash142, 143:

Definition: Acid insoluble ash is a part of total ash insoluble in dilute hydrochloric acid.

This is a test to find out adhering dirt, silica material and sand.

Method:

The ash obtained by the above procedure should be boiled with 25ml of dilute Hcl

for 5 minutes, the obtained insoluble matter is to be collected on Whatman’s filter paper

no. 42 and washed with hot water. The residue to be taken in a crucible, dried and ignited

allowed to cool in a desiccator and weighed. The percentage of acid insoluble ash is

calculated with reference to the air dried drug.144

Applied aspect:

This ash value is used particularly to determine adhering dirt, silica material and

sand.145

3) Determination of Loss on drying

Definition: This is a test to find out loss of moisture content in a given sample on drying.

The remnant material indicates the weight of solid active substance of the given sample.

Procedure146:

The weight of glass–stoppered, shallow weighing bottle that has been dried under

the same conditions to be employed in the determination should be noted. Two grams of

accurately weighed sample should be transferred to the bottle. The bottle should be

covered and weight of the bottle and the contents noted. The sample is to be distributed

evenly as practicable by gentle sidewise shaking to a depth not exceeding 10mm. The

loaded bottle is to be placed in the drying chamber (oven) at 1100C.

50

The stopper should be removed in the chamber; sample should be dried to

constant weight. After drying is completed, the drying chamber is to be opened and the

bottle closed promptly and allowed to cool to room temperature. The percentage of loss

on drying is to be calculated.

Applied aspect:

It determines the amount of volatile matter (i.e. water drying of from the drug).

The moisture content of a drug should be minimized in order to prevent decomposition

either due to chemical change or due to microbial contamination 147.

4) Determination of pH value:

Definition: pH value of an aqueous liquid may be defined as the common logarithm of

the reciprocal of the hydrogen ion concentration expressed in grammes.148

Method:

One percent sample solution is to be prepared by adding 1 gm of sample mixed in

100 ml water and the pH reading to be taken by using a digital pH meter

Applied aspect: -

pH value of a drug signifies the acidic and basic nature of the drug. This helps in

determining the pharmacokinetic property of the drug.

5) Estimation of Arsenic:

Procedure:

Arsenic Trioxide stock Solution: Dissolve 132 mg of Arsenic Trioxide previously dries

at 105 0c for 1 hour accurately weighed in 5 ml of sodium Hydroxide solution in a 1000

ml of volumetric flask. Neutralize the solution with 2 N Sulphuric acid, add 10 ml more

of 2 N Sulphuric acid then add recently boiled and cooled water to volume and mix.

Standard Arsenic solution: Transfer 10 ml of Arsenic trioxide stock solution to a 1000

ml volumetric flask. Add 10 ml of 2 N Sulphuric acid, and then add water to make up

volume and mix. Each ml of standard Arsenic solution contains equivalent of 1 Mg of

Arsenic.

Test Preparation: Add a known quantity of the sample in a muffle furnace at a

temperature not exceeding 4500C.To this add 25 ml of dilute Hydrochloric acid and boil

for 5 min. filter and make up to 50 ml.

51

ATOMIC ABSORPTION SPECTROSCOPY 149 :

Principle:

The principle used in atomic absorption spectroscopy was discovered in 1802

by Wollaston when he observed the "Fraunhoffer lines" or absorption lines in the

spectrum of the sun, yet this principle was only applied in 1955 by an Australian

physicist, Alan Walsh. The principle states that "Matter absorbs light at the same

wavelength at which it emits light". Basically this means that atoms in the ground state

absorb the same radiation as they emit in the excited state. An atom in the ground state

will absorb an amount of energy equal to the energy difference between the energy level

of the electron in the excited state and the energy level that the electron occupies in the

excited state.

In Atomic Absorption Spectroscopy, the sample solution is first vaporized and

atomized in a flame, transforming it to unexcited ground state atoms, which absorb light

at specific wavelengths. A light beam from a lamp whose cathode is made of the element

in question is passed through the flame. Radiation is absorbed, transforming the ground

state atoms to an excited state. The amount of radiation absorbed depends on the amount

of the sample element present. Absorption at a selected wavelength is measured by the

change in light intensity striking the detector and is directly related to the amount of the

element in the sample.

Process: An unknown sample in a solution is dissolved and sprayed finely, in the

presence of suitable conditions, into the flame burner of the atomic absorption

spectrometer. A cathode lamp will emit light to reach these electrons. The lamp must

contain a cathode of the same element within the sample. This is because of the energy

required to excite the similar electrons in the sample, hence enabling concentration to be

determined. Most spectrometers contain a number of different cathode lamps suitable for

various solution samples.

In the lamp, taking in energy excites electrons. They jump to higher energy levels

by taking in a fixed quantum amount of energy. As they fall back down, they emit a fixed

amount of light. This light radiates to the ground atoms in the sample solution, under

specific conditions. These unexcited electrons absorb the light. It is of a fixed

wavelength. As the amount required to excite the electrons in the atoms is fixed,

according to the radiated light, the spectrometer can detect the measure of light absorbed.

In this way, the concentration of the elements can be calculated, as it is directly

proportional to the amount of element present. This is calculated in parts per million

(ppm). The element is detected by an atomic absorption spectrum, by the light intensity

52

emitted by the sample. This is a series of coloured lines on a dark background, depending

on the element, at differing wavelengths.

Applications: This process is employed in both qualitative and quantitative use. AAS is a

rapid method for the former, if only a few elements are being tested. However if many

elements are of interest the process can be too time consuming and uneconomical. The

usual quantitative method brackets the sample's absorption spectrum with that of standard

concentrations to produce a linear calibration curve.

Examples of the applications of AAS include:

• Analysis of water for metals like lead, mercury and cadmium

• Drug testing

• Identification of unknown compositions

• Analysis of rocks on space missions

X-RAY DIFFRACTION 150, 151

Introduction:

X-ray diffraction is a novel and a powerful technique in the elucidation of 3-

dimensional structure, composition of any matter. Structural features can be determined

at molecular and atomic level. It is an important technique for establishing the batch-to-

batch reproducibility of a crystalline form in X-ray Powder diffraction. Random

orientation of a crystal lattice in a powder sample causes the x-rays to scatter in a

reproducible pattern of peak intensifies at distinct angles (θ) relative to the incident beam.

Each diffraction pattern is characteristic of a specific crystalline lattice for a given

compound. Hence X-RD has become invariably indispensable tool in the pharmaceutical

industry in the crackdown of structure.

Discovery: In 1912 Walter Fredrich and C M Paul discovered that if a beam of X-Ray

passes through a crystal or any matter turned in various directions.

Principle: The main principle behind X-RD is the process of scattering of X-rays when it

passes through a matter in crystalline or powder state. The reason for this diffraction

pattern is, X-rays are scattered in various directions by the electrons that form the outer

part of each atom in the matter. If the scattering centers are separated by distances

comparable to the wavelength of the X-rays, then interference between the X-rays

scattered from particular electron centers can occur.

Technique: The technique of X-RD is subjecting the powder matter or crystal matter to

the irradiation by beam of X-Rays and making a record of 3-dimensional diffraction

pattern. In all X-RD procedures the transforming of these diffraction fractions into an

53

image (on photographic film) are involved. For interpretation construction of image is

indispensable with the help of crystallographers and also from modern computers. This

operational process is called ‘Fourier transformation’.

Diffraction as Plane Reflections: The diffraction pattern generated from single crystal

or powder is an array of sharp spots. To explain these parts W.L.Bragg suggested that the

mechanism of X-RD could be cast in terms of reflections from regular, parallel arrays of

planes within a crystal. Only under special conditions would the reflected radiations

interfere constructively and a diffracted beam be observed. The required condition is that

the angle of incidence (θ) of the incident beam obey the following relationship.

Sinθ = n λ/2d

Where lambda (λ) is the wavelength of incident radiation, d is the interplanar

spacing and is an integer constant. This relationship is known as Bragg’s law and theta

(θ) is known as Braggs angle.

Methods:

� Laue method : lambda variable, theta fixed

� Rotating crystal method : lambda fixed, theta partly variable

� Powder method : lambda fixed, theta variable.

Experimental procedure:

1. Choices of radiation – While selecting radiation, the factors to be considered are

Effect of unit cell size, Effect of specific absorption, Relative exposure timings.

2. Sample preparation – Most using method is cylinder technique (rolling with binder

technique). Here the specimen is ground to a uniform size and placed on a glass plate.

A few milligrams of the sample are then mixed with a drop of colliodion until a

homogeneous paste results. The paste then scooped up on a razor blade and rolled in

to a rod shaped specimen with fingers.

3. Powder Camera – Consists of Sample holder, direct beam catcher, Collimator, Screw,

Slider. Basically the method involves diffraction of monochromatic X-rays by a

powder specimen. Each particle in the specimen is a tiny crystal oriented at random

with respect to the incident beam, there is a fair chance that a certain plane will be

currently oriented to reflect the incident beam. Thus every set off planes will be

capable of reflection.

Applications:

54

� Qualitative and quantitative phase analysis of organic and inorganic compounds.

� Determination of grain alignment in polycrystalline materials.

� Determination of exact crystallographic orientation in monocrystalline materials.

� Determination of micro structural properties.

� Determination of ratio of amorphous and crystalline fractions

� Determination of precise lattice parameters.

� Effect of heat treatment and as a tool for characterization of drug formation.

PARTICLE SIZE ANALYSIS 152:

What is particle size? Particles are three-dimensional objects for which three parameters (the length,

breadth and height) are required in order to provide a complete description. As such, it is

not possible to describe a particle using a single number that equates to the particle size.

Most sizing techniques therefore assume that the material being measured is spherical, as

a sphere is the only shape that can be described by a single number (its diameter). This

equivalent sphere approximation is useful in that it simplifies the way particle size

distributions are represented. However, it does mean that different sizing techniques can

produce different results when measuring non-spherical particles.

Particle Size Analysis is an analytical technique by which the distribution of

sizes in a sample of particulate material is measured. Particle size analyzers range from

the historical sieve to modern automated light scattering instruments. The most

appropriate selection for a particular application depends on a number of factors

including the size range of interest, nature of the sample, the information required from

the analysis, sample throughput, and price. Modern light scattering instruments are

becoming the particle analysis method of choice in most industries due to their analysis

speed (approximately 30 seconds), wide size range, ease of use, flexibility,

reproducibility.

Laser diffraction is a non-destructive, non-intrusive method that can be used for

either dry or wet samples. As it derives particle size data using fundamental scientific

principles there is no need for external calibration; well-designed instruments are easy to

set up and run, and require very little maintenance. Additionally, the technique offers: A

wide dynamic measuring range, Flexibility, Generation of volume-based particle size

distributions, Rapid data acquisition, Ease of Verification.

55

The Mastersizer 2000-Model of the instrument. The generic benefits of the

technique of laser diffraction have been exploited and enhanced in the design of the

Mastersizer 2000, which is used worldwide for the analysis of a diverse range of

particles. The Mastersizer has a fully optimized optical design which allows particles in

the size range 0.02 - 2000 micron to be characterized effectively. A range of dispersion

units ensures optimum sample presentation, and switching between units is relatively

easy allowing different samples to be analyzed rapidly, in close succession. Automated,

standard operating procedure (SOP) driven operation delivers a consistent analysis, and

minimizes training requirements, whilst flexible software allows results presentation to

be tailored to the requirements of the customer.

TOXICOLOGY REVIEW Definition: Toxicology is the scientific study of Poisons153. Toxicology (from the Greek words toxicos and logos) is the study of the adverse effects

of chemicals on living organisms. It is the study of symptoms, mechanisms, treatments

and detection of poisoning, especially the poisoning of people.

History 154 Mathieu Orfila is considered to be the modern father of toxicology, having given the

subject its first formal treatment in 1813 in his Traité des poisons, also called Toxicologie

générale.

Theophrastus Phillipus Auroleus Bombastus von Hohenheim (1493 - 1541) (also

referred to as Paracelsus, from his belief that his studies were above or beyond the work

of Celsus - the Roman physician from the first century) is also considered "the father" of

toxicology. He is credited with the classic toxicology maxim, "Alle Dinge sind Gift und

nichts ist ohne Gift; allein die Dosis macht, dass ein Ding kein Gift ist." which translates

as, "All things are poison and nothing is without poison; only the dose makes a thing not

a poison." This is often condensed to: "The dose makes the poison".An even earlier writer

on toxicology was Ibn Wahshiya, who wrote the Book on Poisons in the 9th or 10th

century. The history of poison stretches from before 4500 BC to the present day. Poisons

have been used for many purposes across the span of human existence, most commonly

as weapons, anti-venoms, and medicines. Poison has allowed much progress in branches

of medicine, toxicology, and technology, among other sciences.

56

Authors of the ancient classics were very much aware of this fact and they advocated

great caution while using them.In the description of Visa Vega in human beings. Visa

Vega in animals and birds are also described.

Toxicity 155 is the degree to which a substance is able to damage an exposed

organism. Toxicity can refer to the effect on a whole organism, such as a human,

bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell

(cytotoxicity) or an organ (organotoxicity such as the liver (hepatotoxicity). By

extension, the word may be metaphorically used to describe toxic effects on larger and

more complex groups, such as the family unit or society at large. Toxicity is a function of

solubility. Insoluble compounds as well as the metallic forms often exhibit negligible

toxicity. In some cases, organometallic forms, such as dimethyl mercury and tetraethyl

lead, can be extremely toxic. In other cases, organometallic derivatives are less toxic such

as cobaltocenium cation.

WHY TOXICITY STUDIES?

o To eliminate any frankly poisonous substances from use in clinical trials.

o To determine tolerable dosage range permitting an estimate of the probable

tolerated dose in man.

o To determine theraupeutic index which will indicate to some extent the degree to

which clinical dose schedule may be varied.

o To give some guidance to clinician who will be conducting clinical trials altering

to potential untoward reactions.

o To obtain some clue about mechanism of action of drugs under trial.

Types of toxicity155 There are generally three types of toxic entities; chemical, biological, and physical.

• Chemicals include inorganic substances such as lead, hydrofluoric acid, and

chlorine gas, organic compounds such as methyl alcohol, most medications, and

poisons from living things.

• Biological toxic entities include those bacteria and viruses that are able to induce

disease in living organisms.

Relationship between dose and toxicity156

Toxicology is the study of the relationship between dose and its effects on the

exposed organism. The chief criterion regarding the toxicity of a chemical is the dose, i.e.

the amount of exposure to the substance. Paracelsus, who lived in the 16th century, was

the first person to explain the dose-response relationship of toxic substances.

57

Factors influencing toxicity156 Toxicity of a substance can be affected by many different factors, such as the:

� Pathway of administration (whether the toxin is applied to the skin, ingested,

inhaled, injected),

� The time of exposure (a brief encounter or long term),

� The number of exposures (a single dose or multiple doses over time),

� The physical form of the toxin (solid, liquid, gas),

� The genetic makeup of an individual, an individual's overall health, and many

others.

Toxic metals157 are metals that form poisonous soluble compounds and have no

biological role, i.e. are not essential minerals, or are in the wrong form. Often heavy

metals are thought as synonymous, but lighter metals also have toxicity, as exemplified

by beryllium, and not all heavy metals are particularly toxic and some are even essential

(such as iron). The definition may also include trace elements when considered in

abnormally high, toxic doses. A difference is that there is no beneficial dose for a toxic

metal with no biological role.

Toxic metals sometimes imitate the action of an essential element in the body,

interfering with the metabolic process to cause illness. Many metals, particularly heavy

metals are toxic, but some heavy metals are essential, have a low toxicity, and bismuth is

even non-toxic. Most often the definition includes at least cadmium, lead, mercury and

the radioactive metals. Metalloids (arsenic, polonium) may be included in the definition..

Toxic capacity157 can mean the toxicity of a substance, possibly in relation to a

specific organism and toxic capacity can mean the capacity of an organism, organic

system or ecosystem to contain a toxic substance or a selection of toxic substances (a

compound) without showing signs of poisoning or dying.

A reference dose158 :An estimate, with uncertainty spanning perhaps an order of

magnitude, of a daily oral exposure to the human population (including sensitive

subgroups) that is likely to be without an appreciable risk of deleterious effects during a

lifetime. RfDs are usually derived from animal studies. Animals (typically rats) are dosed

with varying amounts of the substance in question, and the largest dose at which no

effects are observed is identified.

Median lethal dose159, LD50 or LCt 50 (Lethal Concentration & Time) of a toxic

substance is the dose required to kill half the members of a tested population. LD50

figures are frequently used as a general indicator of a substance's acute toxicity. The test

58

was created by J.W. Trevan in 1927. It is being phased out in some jurisdictions in favor

of tests such as the Fixed Dose Procedure., however the concept, and calculation of the

median lethal dose for comparison purposes, continues in wide use. As a measure of

toxicity, LD50 is somewhat unreliable and results may vary greatly between testing

facilities due to factors such as the genetic characteristics of the sample population,

environmental factors and mode of administration.

Dose-Escalation/Dose-ranging:- A trial in which the amount of the drug is either

periodically increased or increased with each new trial arm that is added. Used to

determine how a drug is tolerated animals or humans.

Regulatory authorities : The toxicology studies has its international standards

maintained by WHO that has the protocol from OECD.

OECD GUIDELINES 160:

The Organisation for Economic Co-operation and Development (OECD) (in French:

Organisation de coopération et de développement économiques, OCDE) is an

international organisation of thirty countries that accept the principles of representative

democracy and free-market economy. The OECD is responsible for the OECD

Guidelines for the Testing of Chemicals, a continually-updated document which is ade

facto standard (i.e., soft law).

Acute Oral Toxicity161 : The more traditional test for acute oral toxicity is the oral LD

50 and one test protocol for this is found in OECD Test Guideline 401.

Subacute oral toxicity161: The OECD test guidiline for Sub-acute or short term toxicity

study is under OECD test guideline 407.

59

EXPERIMENTAL REVIEW

Animal testing162 or animal research is the use of non-human animals in scientific

experimentation.

"Tatra Chaturvidha Bhutagramah,Sansvedaja, Jarayuja, Andaja Audbhija Sangnah|

Tatra Purushah Pradhanam, Tasyopakaranam Anyat" !!.163

Man occupies a supreme position among all the living creatures. Hence for

experimental trial, other animals should be utilized as experimental models. While

describing Anna RaksaVidhi, the effects of food contaminated with poison on various

animals and birds are described164. These instances make it clear that, animal

experimentation was undertaken in ancient time also, though antediluvian communication

techniques were different from those of the present day.

In the present study, it is an attempt in determining the toxicity of Shuddha Malla

under the heading of experimental study. Before toxicological studies can begin,

experimental review of animal species used, study types, group size, dose selection and

route of administration are the general issues studied wide infra.

Species selection165: The choice of species for use in toxicological studies is a

compromise between what is required or desired, and what is practical.Naturally when

performing any toxicity study it would be ideal to use a species with the same

metabolism, pharmacokinetics and target organ susceptibility as man.

The Rats-Animals used in the present study:166

The most widely used strains of rat are Sprague-Dawley and Wistar,the latter is a

slightly smaller animal which may offer an advantage for some studies.The animals must

be well examined which is free not only from common diseases but also free from all

pathogenic organisms.Such SPF animals are usually Caesarian derived and then

maintained under special barrier conditions.To minimise the biological variation it is

usual ,when conducting toxicological studies ,to use genetically homogenous SPF

animals.

On arrival in the facility 167:The animals should be marked ,for identification

purposes,and allowed to acclimatise to their new environments and diet.During this time

various pretest measurements can be made.

60

Age Of Rats 167: It is desirable to commence toxicology studies in young rodents,six or

seven weeks old,the animals should be about six weeks old and fully weaned on arrival.

Recording Weight 167: The rats should be weighed on several occasions before the study

begins,and those failing to gain weight at a normal rateor outside a normal weight range

for their age should be rejected.

Steps to be followed before starting the experiment167:

� Adequate number of animals should be available for study.

� Each animal must be individually tested.

� Injuries,abnormalities and lesions should be rejected.

� Before allocated to groups rats must undergo basic haematological and

biochemistry tests.

� Record the weight of all animals.

Behaviour167 : Rats are usually friendly and amenable animals if handled gently,

although there are some strain differences. Rats have a tendency to be nocturnal. Feeding,

drinking and mating all usually occur at night. Their eyesight is poor, and blind rats will

behave as if perfectly normal.

Housing167 : Rats may be kept in metal or plastic cages. If mesh floors are used, care

must be taken to ensure that the mesh is small enough that young animals do not fall

through it. Solid bottomed cages are best and wood shavings, wood chips or paper may

be used as bedding. Cages should be clearly marked, with an individual label for each

animal.

Cages should be arranged on racks in such a way as to distribute animals from

different groups evenly in the various positions in the rack and around the animal room.

All these steps can overcome the ‘animal placement effect’.

Standard diet 167: Rats, like all rodents, are coprophagic. They can be fed adlibitum for

restricted time on a complete pelleted rodent diet, from hoppers suspended above the

floor of the cage. The diet should contain 20-27% protein. Rats will eat 5 g feed per 100 g

body weight daily.

Water167 : Water may be provided by sipper tubesor by automated watering systems. The

water may need to be acidified or chlorinated to reduce contamination. Particularly for

immunocompromised rats. Rats will drink 5ml water per 100 g body weight daily.

Environment 167: Rats are less sensitive to temperature changes than mice, but should be

kept between 21±20 C. The humidity should be 40-70%. A 12- hour light period is

adequate for rats, but, being nocturnal, bright light is deleterious particularly for albino

rats, and results in retinal degeneration. The level should be less than 400 IX or 100 IX

61

for albinos. Photoperiod affects the oestrus cycle, and 12-16h light is best for optimal

breeding.Housing Environment has been observed to influence the toxicity of some

drugs.

Breeding167 : Puberty occurs at 50-60 days, and breeding begins at 3 months when

female weigh 250 g and makes 300 g. They breed until they are 12-18 months old.

Oestrus occurs every 4-5 days. Mating usually occurs at night, and a copulatory plug of

gelatinous material is left in the vagina of 12-24 hours, which then falls out and can be

detected to confirm that mating has occurred. Gestation lasts 21-23 days. A litter of 6-12

pups is born in a shallow nest made by the female.

Growth : Male rats exhibit prolonged growth, and bones do not become fully ossified

until their second year.168

Group Sizes169: The minimum number of animals in treatment groups is dictated by the

regulatory authorities.It may be advisableto to increase the numbers of experimental

animals as there is a desire of accuracy and sensitivity.Normally 6-10 rats in a group is

appropriate.

Route of administration170:

As a general rule drug administration in animals must include the route to be

used in human dosing ,as well,usually,as the oral route.The method of dosing are

dependent upon the circumstances of the study.When rodents are used the drug is usually

given as a suspension or solution by gavage.Dosing by gavage also has its problems.It

requires skill on tha part to avoid dosing accidents.high doses of hypertonic ,irritants can

cause toxicity in the respiratory tract by reflux from the oesophagus to the trachea.Such

problems can be minimised by using suitable catheters.

Dose selection171:

The usual way of conducting the toxicological study,as advocated by the

regulatory agencies, is to use three dose high,medium and low,design with a negative

placebo or vehicle dosed control group.The basic idea is to assess the target organ

toxicity directly and and to determine a threshold effect.By charecterising the toxic

response over an experimental dose range it is possible not only to compare the relative

toxicity of the test agent but also to determine a NOEL (no observed effect

level).Determination of NOEL is important in order to ensure that the dose of drug which

produces the desired pharmacological response is lower than that responsible for causing

any toxicological side effects.

62

In acute,Subacute,and Subchronic studies ,when attempting to identify target

organs of toxicity ,the dose may be so high as to cause death.A dose that causes a 10%

reduction in body weight gains one that causes minimum toxicity.It is usual to include at

least one intermediate dose ranging group,which can provide information on dose-related

effects.An important point about dose selection is that,while animals may receive similar

doses in terms of mg/kg body weight ,pharmacokinetic analysis may demonstrate marked

differences in terms of drug blood concentration and or drug retention time.

Study Protocol :

The present study is carried on two main protocols, design for Acute and Sub-

acute models which are described as following:

Acute toxicity172 describes the adverse effects of a substance which result either

from a single exposure or from multiple exposures in a short space of time . To be

described as acute toxicity, the adverse effects should occur within 14 days of the

administration of the substance.

It is obviously unethical to test for acute (or chronic) toxicity in humans. However,

some information can be gained from investigating accidental human exposures (e.g.

factory accidents). Otherwise, most acute toxicity data comes from animal testing or,

more recently, in vitro testing methods and inference from data on similar substances.

Steps to be observed:

� To establish the maximum tolerated dose.(the highest dose not causing death or

life threatening toxicity) and the minimum lethal dose.

� Single dose studies to establish target organ toxicity.

� Determination of precise LD 50 or median lethal dose.

Type 1173: The group of rats are left untreated for 14 days after a single clinical dose on

first day,during which time they are obsereved daily for the development of clinical

symptoms.On day 15 they are killed and subjected to a thorough examination post

mortem.

Type 2173: To determine the target tissue during the 14 days clinical dose single clinical

dose of test agent should be administered to group of rats for 14 days and observed.It is

a fixed dose procedure. On day 15 they are killed and subjected to a thorough

examination post mortem.

Type 3173: To establish a precise LD 50 the purpose of dose ranging study is fulfilled.To

investigate the toxicity of repeated doses of an agent at dose levels ranging from a very

low multiple of the therapeutic effective dose (somewhere in the range of ED 50 or the

63

anticipated human therapeutic dose range ), up to doses approaching the minimum lethal

dose.The procedures used can vary but usually involve exposing experimental animals to

various concentrations of the drug i.e from a pharmacological dose to minimum lethal

dose or vice versa.The increasing dose levels in a step wise sequence until a maximum

tolerated dose is reached.As the animals tolerate the treatment day by day the dosewill be

raised until the highest dose tolerance is achieved.This is called an ascending phase

maximum tolerated dose (MTD) study.The dead animals are dissected for the

histopathological studies.

In the present study the drug dosage was for single therapeutic dose for one day,single

therapeutic dose for 14 days,dose ranging for 14 days.Thus such a method would provide

information on limiting toxicity and assist in establishing the maximum dose to be used

in subsequent subacute studies.In the rodents three types of toxicity study may be

performed.

All the data obtained from the above studies provide further information on the toxic and

lethal potential of the drug and assist in dose selection for subsequent toxicity studies.

Subacute Toxicity Study174: Also meant as short term toxicity study conducted to screen

for potential adverse effects using rats as surrogates.This study may be of varying

duration generally 1 to 4 weeks or 28 days which has two purposes:

� To observe the possible toxicity for 28 days after administration of a single

therapeutic dose on first day.

� To observe the possible toxicity for 28 days with a single therapeutic dose

administered daily.

The purpose of these studies is an investigation to identify the target organs or systems at

risk .It also gives an account of predictive adverse effects and identify NOAEL..Two

animals must be sacrificed from all groups to evidence such changes.

MEASUREMENTS AND OBSERVATIONS 175 :

Assessment of the behaviour of animals should be carried out informally

on a daily basis before dosing and throughout the study.Rodents should be placed on a

flat surface and their posture ,movements and behaviour observed.They should then be

checked for condition of the fur and for the damaged areas of skin,subcutaneous

swellings or lumps being measured ,abdominal distension etc.,All natural orifices should

be checked for discharges,blockages,,blood,mucus,etc.,the eyes examined for

dullness,dryness,discharges,opacities,pupil diameter,ptosis,exopthalmous etc.,the colour

and consistency of the faeces should be noted and any wetness or soiling of the perinium

64

is recorded.The mouth should be examined for excessive salivation,lumps,cuts etc.Any

breathing abnormalities such as sneezing ,wheezing,rattles of dyspnoea must be recorded.

Body weight176: Changes in the body weight can be a sensitive and important monitor of

health of an animal.Loss of body weight,failure to gain body weight at a normal rate,is

frequently the first indication of the onset of an adverse effect.

LABORATORY PARAMETERS 177:

To provide a valuable clues and indications of the pathological changes

occuring as a result of drug treatment we need to use similar measurements.We are not

involved in making a clinical diagnosis but rather attempting to detect the treatment

related effects,untreated control groups are almost always included in toxicity studies to

provide greater precision in identifying toxicological responses.Clinical chemistry and

haematological measurements can provide can provide useful information on early

toxicological effects,target organ toxicity and reversibility of effects,etc.

Blood Sampling techniques178: For rodents a number of methods can be used.Very

large samples can be taken under general anaesthesia from the venecava,but this

procedure is one from which the animals are not permitted to recover.Retro-orbital

puncture are the widely used methods to obtain the samples.Volumes of upto 2ml can be

obtained from rats.In general blood samples are collected at the beginning during and at

the end of treatment.

Haematological measurements179:

This is to identify the the toxic effects on cellular constituents of blood .To

assess the effects on red cells it is usual to perform RBC,Hb,PCV etc.,Such parameters

provide information on the estimation of the number of circulating red cells,the oxygen –

carrying capacity of the blood and the volume of red cells expressed as a fraction of total

blood volume respectively.

Blood Chemistry180: It is of great importance since they provide early indications of a

toxic dose;assist in identifying target organs toxicity and provide the information of

reversibilty of such effects.

TERMINAL STUDIES 181:

At the end of the treatment,usually 24 hours after the last dose,the

experimental animals are given a final thorough examination for any external

abnormalities and then killed.Rats are usually deeply anaesthetised by exposure to over

dose of diethylether vapour.When it is certain that they are deeply anaesthetized and

insensitive to pain,i.e cessation of the palpebral reflex,they are usually exsanguinated.The

standard selection of organs and tissues are removed from the body cavity and stored in a

65

preserving fluid (fixative) for later histopathological examination.Changes in

histopathological responses if present then changes in organ weights become of

secondary importance.

GUM ACACIA- Vehicle drug and as control.182

This is the dried gummy exudates obtained from the stems and branches of

Acacia Senegal or other African species of Acacia. It has no pharmacological action and

inert. This is used as a suspending agent for the oral administration of the trial drug and

as a control drug in 1% strength..

Controls: A concurrent control group which is identical in every aspect to the treated

groups,except for exposure to the test substance,should be used.

Statistical Methods183, 184,185:

Descriptive statistical analysis has been carried out in the present study.

Results on continuous measurements are presented on Mean ± SD (Min-Max) and results

on categorical measurements are presented in Number (%). Significance is assessed at 5

% level of significance. Analysis of variance has been used to find the significance of

study parameters between the groups, Repeated measures ANOVA has been used to find

the significant changes of parameters with in each group. Percentage of change from

baseline has been computed in the present study to study the pattern changes of toxicity

during the study period.

Analysis of Variance: F test for K Population means

Objective: To test the hypothesis that K samples from K Populations with the same mean.

Limitations: It is assumed that populations are normally distributed and have equal

variance. It is also assumed that samples are independent of each other.

Method. Let the jth sample contain nj elements(j=1,2,…K). Then the total number of

elements is

∑= njN ∑=nj

xijjx.

KN

jxx

S

n

i

−

−=

∑∑−

1

1

2

21

).1(

1

)...(1

1

2

22 −

−=∑

−

K

xjxnj

S

n

i

F=S22/S1

2 Which follows F distribution (K-1, N-K)

66

PHARMACEUTICAL STUDY

This section deals with the shodhana of Malla by Swedana Procedure.

Aims and objectives:

The aim of present study was intended to carry out Shodhana of Malla by a

single Shodhana procedure in Post Graduate pharmacy section of Taranath Govt

Ayurvedic Medical College, Bellary.

The objectives include:

• Selection of raw materials.

• Shodhana of Malla by Swedana procedure.

The materials and methods were selected according to principles of Rasashastra

and practical experience.

Materials:

• Major raw material.

• Other raw materials.

• Yantras and Upayantras.

Major raw material:

The major raw material of the present study is Malla . 500gms of genuine Malla was

collected from the local market fulfilling the Grahya Lakshanas explained in classics.

Other raw materials:

The other raw materials used for the present study are Karavellaka swarasa

(Momordica Charantia), Water, etc.

Yantras: Dolayantra and Khalvayantra were used in the present study.

Upayantras:

Steel vessels, mud pots, weighing machine, Gas stove, Wooden rods, knife,

mixer, Thread, Kora cloth, pH paper, Match sticks, Spoons etc.

Method:

The whole method includes :

Shodhana of Malla by Dola Yantra Swedana procedure.

Methods selected for shodhana in the present study:

• Swedana in Karavellaka swarasa (bitter gourd juice) for 2 yamas (6hrs).

The shodhana was done according to classics, the procedures and observations are

explained under the heading of experiments.

67

EXPERIMENT :1.

Shodhana of Malla

Date of commencement : 06.09.2007

Date of completion : 06.09.2007

Reference : R T 11/136.

Materials : Malla: 1–50gms in 1st pot.

Malla: 2-50gms in 2nd pot.

Karavellaka Swarasa 3450 ml to Malla: 1

Karavellaka Swarasa 3650 ml to Malla: 2

Equipments : Mud pot, gas stove, steel vessel, wooden rod, thread,

Weighing machine, mixer.

Principle : Swedana in Dola yantra containing Karavellaka Swarasa.

Procedure: Preparatory procedure followed were two in number.

A. Extraction of Karavellaka Swarasa:

About 111/2 kgs of Karavellaka fruits were taken, cut at the center and

removed the seeds and juice was extracted by using mixer, filtered through a clean cloth

and measured, which was about 9 ltrs.

B. Preparation of Dola yantra:

A wide mouthed mud pot was taken, a wooden rod was placed over the rim

horizontally. Half of the vessel was filled with the Karavellaka Swarasa which was1ltr. A

clean, white, square shaped kora cloth measuring 10 inches in length and 10 inches in

breadth was taken. 50gm of small pieces of Malla were placed at the center and a pottali

was made and strong thread was used to tie the pottali. This pottali was suspended again

by tying to the wooden rod placed on the rim of vessel containing Karavellaka Swarasa.

Main procedure:

Properly prepared Dola yantra was kept on gas stove for heating. Mild

heat (Mrudvagni) was given for 6hrs(2 yamas). Karavellaka Swarasa was added as and

when required to maintain the minimum liquid level. After 6hrs(2 yamas), heating was

stopped and the contents were allowed to self-cooling. Then the pottali was removed

from the liquid and washed thoroughly with hot water. Then the pottali was opened and

68

Malla was collected and washed thoroughly with hot water and kept spread for drying on

a clean white cloth.

Observations::

• Light Green colored swarasa turned to brownish yellow color at the end of the

procedure.

• At the end consistency of liquid mixture was thick when compared to fresh

condition.

• 1ltr of Karavellaka swarasa was sufficient to immerse the pottali.

• For every 30-40 mins about 100-150 ml 0f swarasa was added as the level

reduced.

• The Karavellaka swarasa required for whole process was 4.5 ltrs.

• A characteristic smell of Karavellaka was felt during initial stage of boiling.

• Greenish yellow scum had started to accumulate at the end of half an hour.

• The Greenish yellow scum was found over the pottali at the end of swedana.

• The solution became more acidic.

• Swarasa contained minute particles which tend to sediment with the time.

• Before subjecting to swedana, the color of Malla was white and after subjecting to

swedana also there is no change in color.

• After complete drying, the Malla was white in color with slight shining.

• There was a minimum change in weight of malla.

• Temperature range 900C -970C.

Table No 8: Showing the observation during Malla Shodhana

Particulars Before Shodhana After Shodhana

Colour of Karavellaka

Swarasa Light green Brownish Green

Consistency of swarasa Liquid and Clear

Increased with suspending

Particles

Odour of the Swarasa Natural fresh odour of

swarasa Very Obsessive odour

69

pH of swarasa 6.84 (Acidic) 6.72 (More acidic)

Colour of Malla White White

Appearance Shiny/glassy Opaque dull.

Weight of Malla 1)50gm

2)50gm

1)46gm

2)47gm

Precautions:

• Sufficient quantity of Karavellaka swarasa was taken to immerse the pottali

completely.

• The liquid in the vessel is maintained to immerse the pottali throughout the

procedure.

• The liquid was added according to requirement.

• The pottali was tied properly to avoid spilling of small pieces of Malla (from

pottali).

• The pottali was maintained in such a height that it did not touch the bottom of the

vessel.

• Mild heat was maintained throughout the process.

Results:

Weight of Malla before shodhana : 1st Pot - 50gm, 2nd Pot- 50gm.

Weight of Malla after shodhana : 1st Pot- 46gm, 2nd Pot- 47 gm

Loss of weight : 1st Pot- 4gm., 2nd Pot- 3 gm.

70

ANALYTICAL STUDY

Drug analysis consists in the estimation of purity and quality for drugs used

in the Pharmaceutical preparations. The drugs that are manufactured should be

understood well and vividly interpreted in the light of modern chemistry. A quality

control program for Pharmaceutical industries involves, batch to batch uniformity of the

products ensuring that the final product posses desired characteristics of identity, purity,

potency, uniformity, safety, efficacy and stability with in established levels which meet

all the legal, professional and company standards. When these things applied to

Ayurvedic formulations, helps in standardizing the drug in total, for better understanding

& interpretation of Physico-chemical changes occurring in processing’s and also ensure

the safety and efficacy of the drug.

In present study analytical study was done for the following two samples i.e.

• Ashuddha Malla

• Shuddha Malla.

Aims and Objectives:

• To analyze physicochemical properties of Ashuddha Malla and Shuddha Malla.

• To carryout quantitative estimation of Arsenic (As %), in Ashuddha Malla and

Shuddha Malla.

• To carryout X-Ray diffraction studies on Ashuddha Malla and Shuddha Malla.

Materials and methods:

Materials:

• Ancient parameters were conducted at P.G Dept. of Rasashastra, T.G.A.M.C.,

Bellary.

• Modern Physical and chemical tests were conducted at Ganesh Consultancy and

analytical services, Mysore.

• An X-Ray diffraction study was conducted at Indian Institute of Science,

Bangalore.

71

Methods:

Physical tests:

Ancient parameters:

The ancient parameters such as Varna, Sparsha, Gandha were studied at Post

Graduate Department Rasa Shastra, T.G.A.M.C. Bellary. Following results were

obtained.

Varna : White. (Shwetha)

Sparsha : Smooth, fine (mrudu).

Gandha : Odorless. (nirgandha)

Modern Parameters:

The physical tests of two samples of Malla i.e. Ashuddha Malla and Shuddha

Malla were carried out at Ganesh Consultancy,Mysore. The tests included Organoleptic

Characters, Estimation of Total Ash, Determination of Acid insoluble Ash, percentage of

Arsenic, Determination of loss on Drying at 1100C, loss on ignition and pH.

Table No 9: Showing the Organoleptic Characters of two samples of Malla.

Determination of Ash value:

Materials:

1. Silica crucible.

2. Electronic weighing machine.

3. Electric furnace.

4. 2gms of two samples of Malla each i.e. Ashuddha Malla and Shuddha Malla

Procedure: Two grams of accurately weighed, grounded two samples of Malla were

taken separately and transferred to the cleaned, dried and weighed Silica crucible,

previously ignited and weighed. Scatter the ground dry in a fine even layer on the bottom

of the crucible. Incinerate by gradually increasing the heat not exceeding dull red heat

(4500C) until free from carbon. Cool and weigh. The ash value of two samples of Malla

was calculated with reference to the air-dried drug.

Results:The percentages of total Ash value of two samples of Malla are as follows;

DRAVYA VARNA SPARSHA GANDHA

Ashuddha Malla Pure White Smooth, fine

Odorless

Shuddha Malla Dull White Smooth, fine Odorless

72

� Ashuddha Malla : 8.92%

� Shuddha Malla : 8.86%

Determination of Acid Insoluble Ash:

Materials:

1. Silica crucible.

2. Burner.

3. Whatman filter paper.

4. Electronic weighing Machine.

5. Diluted Hcl – 25 ml.

6. Conical flask.

Procedure: The Ash obtained by above procedure was boiled for 5 minutes with 25 ml

of dilute Hcl. It was filtered through Whatman filter paper and was washed with water.

The residue was dried, ignited in a crucible and was allowed to cool. Acid insoluble ash

was collected and the dry weight of acid insoluble ash was estimated with reference to

the air-dried drug.

Results: The percentages of Acid Insoluble Ash value of two samples of Malla are as

follows;

� Ashuddha Malla : 0.77 %

� Shuddha Malla : 0.48 %

Determination of Loss on drying at 1100C:

Materials:

1. Silica crucible.

2. Electronic weighing machine.

3. Electronic air oven.

4. 2gms of three samples of Malla each i.e. Ashuddha Malla and Shuddha Malla.

Procedure: Two grams of two samples of Malla each i.e. Ashuddha Malla and Shuddha

Malla were taken separately in a silica crucible. The weight (W1) of the crucible along

with two grams of above said samples was noted. The samples were then dried in a hot

air oven at 1100C till a constant weight is obtained and the dry weight (W2) was

obtained. The percentage of loss in weight on drying was estimated by difference in

weights (W1-W2).

Results:

73

The percentage of Loss on Drying at 1100 C of the two samples of

Malla are as follows;

Ashuddha Malla : 0. 09%

Shuddha Malla : 0.12 %

Determination of loss on ignition:Weigh a silica crucible previously ignited for one

hour at a temperature not exceeding 5000c. and cool in desiccators. Transfer to the

crucible accurately weighed sample of Shuddha Malla. Weigh the crucible accurately.

Place the loaded crucible in the muffle furnace and ignite the crucible to 5000c, until

constant weight is indicated. Calculate loss on ignition with reference to the air dried

drug.

Result: The percentage of loss on ignition of the two samples of Malla is as follows,

Ashuddha Malla : 82.84 %

Shuddha Malla : 81.39 %

Determination of water soluble ash:Boil the ash for five minutes with 25 ml of water;

Collect the insoluble or matter in a Gooch crucible,ashless filter paper,wash with hot

water,and ignite for fifteen minutes at a temperature not exceeding 450°C .Substract the

weight of the insoluble matter from the weight of the ash,the difference in weight

represents the water soluble ash.Calculate the percentage of water soluble ash.Calculate

the percentage of water soluble ash with reference to air dried drug.

Results: Water soluble ash of the two samples is as follows,

Ashuddha Malla : 0.04 %

Shuddha Malla : 0.05 %

Determination of pH Value:

Materials:

1) Digital pH meter.

2) Water – 100 ml.

3) Two samples of Malla i.e. Ashuddha Malla and Shuddha Malla:

Procedure:1% of the two samples of Malla each i.e Ashuddha Malla and Shuddha Malla

solution was prepared (1 gram in 100 ml water) and the pH reading was taken using a

Digital pH meter.

Results:

pH of the two samples of Malla i.e. Ashuddha Malla and Shuddha Malla;

Ashuddha Malla : 6.98

74

Shuddha Malla : 7.08

Qualitative and Quantitative chemical analysis: Qualitative and quantitative chemical

analysis of the two samples of present study was taken up at Ganesh Consultancy

Services,Mysore.

Estimation of Arsenic:

Methodology: Instrumental method using atomic absorption spectroscopy.

Materials: Atomic Absorption Spectrophotometer with a graphite tube cell.

Make: Analytik Jena AAS, Germany.

Model: Nov AA 4OOG. Sample: Ashuddha Malla & Shuddha Malla each 3 gms. Parameters: Wavelength - 193.7hm

Drying temperature - 1100C.

Thermal decomposition (Phyrolysis) - 11000C.

Atomization-2,2000C

Working range - 10ppb-50ppb.

Method: Weigh 3gm of sample in silica crucible and ashed at 5000C in a Muffle furnace,

after cooling and add 5ml of con. hydrochloric acid. Bril, cool, filter and make up the

volume to 100ml. Further dilutions done, if required to attain the working range of the

instrument.Optimise the response of the instrument for wavelength setting etc. Atomize

blank to get Zero absorption. then atomize the Standards (minimum 3 standards) and

note down the absorption. Then atomise the sample to get absorption value of the

standard against concentration in Kg/Litre of Arsenic. Locate the point of the sample

absorption and calculation of the concentration of Arsenic in the sample.

Calculation:

Percentage of Arsenic: C X V X 100 109 M

Where, C: Concentration of Arsenic in Mg/litre in the final Solution V: Volume in ml of the final Solution. M: Mass in gram of the sample in the final Solution. Results: The quantitative & qualitative estimation of the Arsenic (As%) of the two

samples of Malla i.e.Ashuddha Malla and Shuddha Malla are as follows;

� Ashuddha Malla : 69.72%

� Shuddha Malla :74.22%

75

PARTICLE SIZE: -

Laser Particle Size Analysis consists in measuring the size of particles of the

sample using the diffraction and diffusion of a laser beam. During the laser diffraction

measurement, particles are passed through a focused laser beam. These particles scatter

light at an angle that is inversely proportional to their size. The angular intensity of the

scattered light is then measured by a series of photosensitive detectors. The map of

scattering intensity versus angle is the primary source of information used to calculate the

particle size.

The Mie scattering model accurately predicts the scattering of particles. Mie

Theory allows for primary scattering from the surface of the particle, with the intensity

predicted by the refractive index difference between the particle and the dispersion

medium. It also predicts the secondary scattering caused by light refraction within the

particle .

The particle size of the drugs both Shuddha Malla and Ashuddha Malla was

analysed at Dept.of Materials engineering, IISc ,Bangalore using Model Malvern’s

Particle Size analyzer 2000 for Laser diffraction method.

Table 10: Showing the Summary of particle size analysis.

Volume Under % Shuddha Malla

Size(µm)

Volume

Under %

Ashuddha

Malla Size(µm)

0.04 0.06 0.02 0.06

1.34 0.13 1.65 0.15

10.35 0.36 11.81 0.31

48.78 41.43 48.64 22.49

94.17 258.95 94.78 76.32

100.00 477.01 100.00 120.67

The d50 (50%) of particle size of Shuddha Malla is : 43.32µm.

The d50 (50%) of particle size of Ashuddha Malla is : 23.67µm.

76

X-Ray Diffraction (XRD)

XRD of all samples were carried out at Indian Institute Of Science,Bangalore.

Analytical procedure of XRD:

For the present study the X-ray analysis of two samples were carried out using

JEOL X-ray diffractometer. The machine is equipped with Cu K-alpha (Lambda-1.5406)

radiation and graphite monochromator operated at 40 kV / 30 mA). All samples were

very well grounded to 200 meshes and air-dried. The X-ray diffractometer scans was

made on randomly oriented samples from 3-65° 2-theta (d=29.42 to 1.43 angstrom) with

a step size of 0.02° and 1 second time per step.

The 2-theta value and intensity of the peak (counts) are represented on X and Y-

axis respectively. Please note that higher the value of counts represents higher the

crystallanity of the phase. The crystallanity varies sample-to-sample and experimental

conditions. For identification of each phase, minimum 5 strong peaks were chosen and

compared with standard JCPDS. The standard JCPDS card number, identified phase and

composition are also given.

X-Ray diffraction study:

Table No 11: Shows the chemical composition of 2 samples confirmed after

identification of d-identified, d-standard peak values, 2 theta standard and 2 theta

identified peak values.

Sample :1 Ashuddha Malla. Sample : 2 Shuddha Malla.

Highest

Counts

Standard

Intensity

Relative

Intensity

Sample :1

D

Standard

Peak

Values

D

Identified

Peak

Values

2 theta

standard

2 theta

identified

6858 100% 100% 3.195 3.211 27.902° 27.779°

1885 27% 27% 1.9570 2.55 46.358° 35.198°

1426 21% 21% 1.6700 2.78 54.935° 32.203°

997 17% 15% 2.1320 1.963 46.358° 46.244°

689 10% 10% 1.3530 1.674 54.935° 54.84°

Sample:2

4771 100% 100% 3.195 3.192 27.902° 27.948°

1498 28% 31% 2.7680 2.538 32.315° 35.361°

932 21% 20% 1.6700 2.766 54.935° 32.366°

856 17% 18% 2.1320 1.957 42.359° 46.403°

654 12%

14% 2.2620 1.551 39.818° 59.605°

Crystal Structure of As2 O3 : Face centered cubic.

77

Foot Note 1) Ashuddha Malla :

Totally 25 peaks were identified in the sample from 30-840 2-theta at x-axis

and from 0-8000 counts on y-axis. (d =6.453 to 1.162 angstrom). In this 5 peaks were

chosen as strong peaks with their relative intensities with standard JCPDS. 1st peak was

considered as significant at 27.779°, d observed was 3.211 compared to 3.195 with

relative intensity of 100%, 2nd peak was considered at 35.198°, d observed was 2.55

compared to standard d 1.9570 with relative intensity of 27%. 3rd peak was considered at

32.203°, d identified was 2.78 compared to 1.6700 with relative intensity of 21%. 4the

Sample :1:Ashuddha Malla

• JCPDS card no:04-566

• Composition: Arsenolite,As2O3

• Scan date:28.08.2008

• Ref:Swanson,Tatge,Natl.Bur.Stand (U.S),Circ.539,1.51 (1953)

• Color:Colorless

Pattern at 26O C.A Mallinckrodt Sample. It was recrystallised by

sublimation.Spectroscopic analysis shows Fe,Mg,Sb,Si 0.001 to 0.01% each.Ca and Pb

trace.Isostructural with senarmontite.Sb2 O3.

• As2O3 type.Also called Arsenic glass,WhiteArsenic,.PSC:cF80.

• Molecuar wt:197.84

• Unit Cell Volume:1358.41

78

peak was considered at 46.244°, d identified was 1.963 compared to Standard 2.1320

only, with relative intensity of 15%. 5th peak was considered at 54.84°, d identified was

1.674 compared to 1.3530 with relative intensity of 10%.

Sample 2: Shuddha Malla

• JCPDS card no:07-400

• Composition:Arsenolite,As2O3

• Scan date:28.08.08

• Ref:Calculated from ICSD using POWD-12++.(1977)

• Ref: Pertlik, F.,Czech, JPhyscs..28.170 (1978)

• As2O3 type.PSC:cF80

• Molecular wt:197.84

• Unit Cell Volume:1358.04

Foot Note 2):- Shuddha Malla

Totally 25 peaks were identified in the sample from 30-840 2-theta at x-axis

and from 0-8000 counts on y-axis. (d =6.453 to 1.162 angstrom). In this 5 peaks were

chosen as strong peaks with their relative intensities with standard JCPDS. 1st peak was

considered as significant at 27.779°, d identified was 3.211 compared to 3.195 with

relative intensity of 100%, 2nd peak was considered at 35.198°, d identified was 2.55

compared to standard d 1.9570 with relative intensity of 27%. 3rd peak was considered at

32.203°, d identified was 2.78 compared to 1.6700 with relative intensity of 21%. 4th

peak was considered at 46.244°, d identified was 1.963 compared to Standard 2.1320

only, with relative intensity of 15%. 5th peak was considered at 54.84°, d identified was

1.674 compared to 1.3530 with relative intensity of 10%.

79

EXPERIMENTAL STUDY

Knowledge of the mode of action of a drug obviously greatly enhances prediction

from animal studies of what will happen in man. Mineralo-metallic forms that are

developed into drugs must have therapeutic efficacy and be safe. Therefore, in the

development of drug it is essential to select drugs that have a margin of safety between

the dose that produces the desired (therapeutic) and the dose that produces undesired

(toxic) effects. The margin of safety, for some drugs is small, and some people

intentionally overdose themselves. As a result, toxic effects of drugs often are observed.

The experimental study should be well planned and various inferences are to be

drawn with respect to therapeutic dosage, route of administration and safety. The

evidences collected out of the experimental study should be accurate and beyond the bias

and errors so as to get reproducibility and hence accountability. Thus Rasadravyas and

Ayurvedic formulations can be brought into GMP as well as GCP.

The task of animal experiments is to minimize the possibility

of missing the useful drug and to obtain maximum information from relatively fewer

animals. Animal experimentation for screening of drugs has a number of limitations like

interspecies variation still the need of the hour is Safety.

The present study is one such attempt in determining the toxicity

profile, acute and sub-acute of the drug Shuddha Malla which is a potential toxicophore

,simultaneously is one of best drugs described therapeutically in recommended doses

classically as seen in review. Such Pre-clinical database is not been obtained until now.

Thus its time for proving the safety related studies through toxicology.

Locale of the Study:-

The toxicological studies was conducted according to the WHO

protocols for screening the drug Malla (As2O3) for toxicology in the Department of

Pharmacology and Toxicology, Veterinary College, Karnataka Veterinary, Animal and

Fisheries Sciences University, Hebbal, Bangalore -560024.

80

ACUTE STUDY

The initial acute study is generally a single dose and a single dose with 14 days of short

term repeated dose study observation186. It is necessary to run a more comprehensive

range of dose levels in short term repeated dose study which increases the chances of

defining the dose response188.

The present study is for 14 days period in which Shuddha Malla is the test drug.

Study Start Date: 5th October 2007

Study End Date: 3rd November 2007

Objective

� To screen the probable toxicity of the drug Shuddha Malla on albino rats with a

single dose.

� To screen the potential toxicity of the drug Ashuddha Malla on albino rats with a

single dose.

� To screen the probable toxicity of the drug Shuddha Malla on albino rats with

repeated doses for 14 days.

� To screen the potential toxicity of the drug Ashuddha Malla on albino rats with

repeated doses for 14 days

� To establish the median lethal dose of drug Shuddha Malla.

� To establish the median lethal dose of the drug Ashuddha Malla.

Materials:

Animals : Wistar Albino rats (Weighing 150-200 gms).

Drug and Chemicals :

• Karavellaka Shodhita Shuddha Malla.

• Ashuddha Malla (raw sample before shodhana)

• EDTA (Disodium ethylene diamine tetra acetic

acid)(MERCK)

• Diethyl ether

• NBF 10% (neutral buffered formalin)

(40% formalin-100ml, distilled water-900ml, Sodium

phosphate monobasic-4 gm, Sodium phosphate dibasic-65gm)

• Gum acacia.

81

Equipments : Digital animal weighing balance

Milligram digital weighing machine.

Centrifuging machine

Serum biochemical semi auto- analyzer

Sterilizing oven.

Other accessories: Tuberculin syringe (1ml).

2ml and 5ml syringe.

Gavage needle for intra gastric or oral administration.

Surgical Set, tissue paper roll, cotton roll

Storage Vials 5ml pp, Capillary tube 2mm bore

Micro centrifuge tube 2ml pp

Plastic containers.

Surgical gloves.

Methods:

Pre procedure: 1) Selection of animal species:

Healthy Albino Rats of either sex weighing 150-200gms were randomly selected

and used in the present study. The animals were obtained from the Central animal

House,Indian Institute of Science, Bangalore.

2) Housing and feeding conditions:

Animals were kept in polypropylene cages with paddy husk bedding. Sex

distribution was maintained equal in all age groups. The temperature in the experimental

room was around 240C. Lighting was natural, the sequence being 12 hours dark, 12 hours

light. They were provided standard food pellets procured from Amruth Feeds,B’lore and

water ad libitum.

The experimental procedures were carried out in accordance with the ethical

guidelines for animals proposed by the CPCSEA , Government of India. The approval

of the ethical committee, Karnataka Veterinary, Animal and Fisheries Sciences

University, Hebbal, Bangalore was obtained before starting the experiments.

82

3) Grouping:

Before grouping the inclusion and exclusion criteria was fulfilled.

Inclusive criteria:

1) Adult healthy albino rats from both the genders.

2) Rat weighing 150-200gms

3) Albino rats between 90-120 days were included.

Exclusive criteria :

1) Unhealthy albino rats

2) Weight below 150gms and above 200gms

3) Albino rats of below 90 days and above 120 days were

excluded.

The remaining animals were randomly selected, 56 animals were equally divided

into seven groups of eight animals each. In each group the animals were marked with

numbers to permit individual identification. Each group of 8 animals was kept in separate

polypropylene cages, for one week prior to dosing to allow acclimatization to laboratory

conditions.

4) Examination of the animals prior to the experiment:

• Heart rate was counted as number of beats/minute by feeling the heart rate by

thumb.

• Respiratory rate was counted as number of inspiration and expirations/minute

observing the movement of abdomen.

5) Fixation and preparation of rat dose:

The normal human adult dose of Shuddha Malla is 1/120-1/30 Ratti, which is

equal to 1mg-4mg. This was converted into animal dose based on Paget and Barner’s

surface area ratio which works out to be 0.36mg /kg body weight.

i.e. Rat dose / kg body wt. = 0.018 x Human dose x 5

= 0.018 x 1/30 Ratti x 5

= 0.018 x 4mg x 5

Therefore Rat dose of Shuddha Malla = 0.36 mg / kg body weight.

83

Similarly, total daily dose of Ashuddha Malla is taken as 0.36mg/kg body weight.

Vehicle for Administration of Drug:

As the drugs are insoluble, the gum acacia 2% solution (by mixing 2 gms of gum

acacia in 100 ml of distilled water) is taken as the vehicle and the same as control.

The 2% gum acacia is calculated for rat dose of 5 ml /kg body weight.

Procedure:

Administration of Drugs:

Drug was administered through intra gastric tube using 2 ml syringe fitted with 18

gauze needle made of steel provided with 5 number infant feeding tube to avoid injury to

the rats during drug administration. Prescribed dose of suspended drug was loaded in

syringe and the tube was inserted into the oesophagus. After confirming that the tube was

inside the oesophagus, drug was pushed slowly to reach the gastrum.

Table No. 12: Shows drugs according to groups.

Group Number of Rats Drug Purpose Group I 8 2% gum acacia To serve as control

Group II

8

Shuddha Malla

single dose only on

1st day &

observation for 14

days.

To serve as test trial

group and observe

for the single

therapeutic drug

effect.

Group III

8

Shuddha Malla

single dose for 14

days daily.

To serve as test

trial group and

observe for the

single dose daily

until 14 days.

Shuddha Malla in To serve as test trial

84

Group IV 8 escalated doses for

14 days.

group for evaluating

median lethal dose.

Group V

8

Ashuddha Malla

single dose only on

1st day &

observation for 14

days.

To serve as test trial

group and observe

for the single

therapeutic drug

effect.

Group VI

8

Ashuddha Malla

single dose for 14

days daily.

To serve as test

trial group and

observe for the

single dose daily

until 14 days.

Group VII

8

Ashuddha Malla in

escalated doses for

14 days.

To serve as test trial

group for evaluating

median lethal dose.

Foot note:-The grouping was done randomly from both the genders to prevent bias.

Table 13 :Shows the weight and administered dose accordingly in each

group for 14 days.

Group Weight of the Rat Calculation of dose Administered dose

Group I ±175 5 x 175 1000

0.75 ml

Group II ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group III ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group V ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group VI ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group IV and VII are administered doses in increasing range by multiples of 5

each day to the therapeutic dose173. Therefore the following table shows the doses given

to these groups for 14 days schedule in mg/kg body weight which was converted to ml

with gum acacia solution.

85

Table 14: Shows the escalated doses of the test drug charted accordingly

to Group IV & VII. (Ref: Fixed dose Procedure) 189

Group

IV &

VII

Day

1

Day

2

Day

3

Day

4

Day

5

Day

6

Day

7

Day

8

Day

9

Day

10

Day

11

Day

12

Day

13

Day

14

Malla 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4

Gum

acacia

soln

0.63

ml

3.1

ml

6.3

ml

2.1

ml

1.4

ml

1.8

ml

2.1

ml

1.2

ml

1.4

ml

1.6

ml

1.8

ml

1.9

ml

2.1

ml

2.0

ml

Foot note:

As the concentration of the drug increased the volume of the vehicle decreased because the gastric capacity of the rats is only 4-5 ml. Both the respective groups received as per the dose schedule above. These two groups are kept under observation to know the median lethal dose. Main Study Procedure:

� The animals from all groups were administered their respective drug dosage

everyday as shown in the above tables.

� General clinical observations on feed and water consumption, posture, gait, and

social behaviour. The health condition of the all the animals was recorded. All the

animals were observed thrice daily for morbidity and mortality.

Method: Rats were placed on the flat surface to observe the posture, movements

and behaviour.Gently feel from head to tail to check the condition of the fur and

damaged areas of skin, areas of tenderness. All natural orifices were checked for

any discharges, blockages, blood, mucus etc., the color and consistency of the

faeces.The mouth examined for excessive salivation, breathing abnormalities etc.

� Body weight recorded: The body weight of all the rats are recorded for every 3rd

day to know the significant changes.

� The rats from Group I, II, III, V and VI were sacrificed on 14th day to trace out

any histopathological changes.

� Group IV and group VII are the groups with escalated doses of Shuddha Malla

and Ashuddha Malla respectively which were under observation for morbidity

86

and mortality. Autopsy of such dead animals was carried out and preserved the

organs for histopathological study.

The following table gives the account of dead and alive rats in particular doses.

Table 15: Showing the day and number of rats dead in Group IV .

Group

IV

Day

1

Day

2

Day

3

Day

4

Day

5

Day

6

Day

7

Day

8

Day

9

Day

10

Day

11

Day

12

Day

13

Day

14

Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4

R1 A A A A A A A A A A D - - -

R2 A A A A A A A A A A D - - -

R3 A A A A A A A A D - - - - -

R4 A A A A A A A A A A D - - -

R5 A A A A A A A A A A A A A A

R6 A A A A A A A A D - - - - -

R7 A A A A A A A A A A D - - -

R8 A A A A A A A A A D - - - -

Foot note: A –alive, D-dead, R-rat number. In Group IV maximum mortality of rats was on 11th day at dose of 18 mg which is the maximum tolerated dose. The minimum tolerated dose is 14.4 mg on 9th day. The median lethal dose is calculated on this base189. Table 16: Showing the day and number of rats dead in group VII.

Group

IV

Day

1

Day

2

Day

3

Day

4

Day

5

Day

6

Day

7

Day

8

Day

9

Day

10

Day

11

Day

12

Day

13

Day

14

Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4

R1 A A A A A A D - - - - - - -

R2 A A A A A A A A D - - - - -

R3 A A A A A A A A D - - - - -

R4 A A A A A A A A D - - - - -

R5 A A A A A A A D - - - - - -

R6 A A A A A A A D - - - - - -

R7 A A A A A A A A D - - - - -

R8 A A A A A A A A D - - - - -

87

Foot note: A –alive, D-dead, R-rat number. In group VII maximum mortality of rats was seen on 9th day i.e. at the dose of 14.4 mg which is the maximum tolerated dose. The minimum tolerated dose is 10.8 mg on 7th day. The median lethal dose is calculated on this base. Collection of blood samples:- Blood of approximately 4 ml volume was drawn from

the retro-orbital plexus using a capillary tube under ether anaesthesia,after an overnight

fast. Blood samples, mixed with disodium ethylene diamine tetra acetic acid (EDTA) as

were used for haematological measurements. Serum samples were separated by

centrifugation at 5000 rpm for 20 min, and then were stored at - 80°C until analysis for

serum biochemical measurements.

� Investigations:

1. Haematology:-

The following haematological parameters were determined using blood

samples collected on day 0,7th and 14th day using microhaematocrit capillary

tubes.

1. Haemoglobin levels(Hb)

2. Total Leucocyte Count (TC)

3. Diffrential Count.(DC)

4. Packed Cell Volume.(PCV)

5. Red Blood Corpuscles (RBC)

2. Serum biochemical parameters:-

Serum biochemical parameters were estimated from the serum samples

collected from the animals on Day 0,7th and 14th day using a semi automatic auto

analyzer and ready to use kits (Swemed diagnostics, Bangalore).The following

humoral parameters were estimated :-

1. Serum Urea.

2. Serum Creatinine

3. SGOT (Serum glutamic oxalacetate transaminase)/AST

4. SGPT (Serum glutamic pyruvate transaminase)/ALT

� COLLECTION OF ORGANS FOR HISTOPATHOLOGICAL STUDIES: -

At the end of experimental period of 14 days, the animals from all the

groups were sacrificed except group IV & VII and observed for gross lesions of

internal organs. The main organs like Heart,Brain,Liver,Kidney,Lungs,Spleen and

88

Gastric mucosa were collected separating it from adhering tissues using saline and

collected by placing on blotting paper and gently pressed to remove excess of

saline.The tissues were also collected from the rats which are dead due to escalated

doses on particular days post-mortem however,severly autolysed were only

necropsied and inspected for obvious changes.

Fixation :

The tissues were excised out immediately after sacrificing, cleaned of

extraneous tissue cut into pieces of 3-5 mm thickness and transferred to 10% formalin

solution. The tissues were allowed to remain in it till they are taken up for processing.

Tissue Processing :

Tissues were thoroughly washed by placing them under running tap water and

placed in 70% alcohol. The tissues were subjected to dehydration, clearing and paraffin

infiltration by passing them through 80, 90 and 95% alcohol (2 changes), isopropyl

alcohol, acetone (2 changes) chloroform (3 changes) paraffin (2 changes) (3 each). Next

the tissues were embedded in paraffin to prepare tissue block in paraffin. Tissue blocks

were fixed to metal object holder after trimming them to suitable size.

Section Cutting:

The tissue sections (5 µm) were cut with help of a Spencer type rotary

microtome and floated in a water bath between 40-45°C. Then they were mounted on

clean glass slides with a drop of Mayer’s egg albumin, dried on hot plate at about 50°C

for 30 min.

Staining Procedure (Haematoxylin Eosin Stain) :

The sections were stained by serially placing them in xylol, acetone, 95%

alcohol, running water, haematoxylin stain, running water again, eosin solution 95%.

alcohol (3 changes), acetone (2 changes), xylol - 2 changes and mounted with D.P.X.

The slides were viewed under a microscope at various magnifications to note down the

microscopic features.

STATISTICAL ANALYSIS:- The data generated from the experimental study was

subjected to one- way ANOVA (Analysis of Variance) by statistical analysis.

89

SUB-ACUTE STUDY Repeated dose toxicity study are conducted to screen for the potential adverse

effects of the drug chemical using lab animals as surrogates for a target species, most

often the human. The study duration is for about 4 weeks187.A 28 day study can produce

more valuable information than a 14 day study because of the long term exposure

increases the probability of detecting more slowly developing adverse effects, assuming

the dose range tested is similar188.

The present study is for 28 days period in which Shuddha Malla is the test drug.

Objectives:

� To screen the probable adverse affects of the drug Shuddha Malla on albino rats.

� To screen the probable adverse affects of the drug Ashuddha Malla on albino rats.

� Identify the target organs.

� Determine need for specialized endpoints to be assessed in long-term studies.

Materials:

Animals : Wistar Albino rats (Weighing 150-200 gms).

Drug and Chemicals , Equipments , Other accessories: Same as acute study.

Methods:

Pre procedure: Selection of animal species, housing and feeding conditions, Inclusive

exclusive criteria and Examination of the animals prior to the experiment are all similar

to Acute Studies.

Grouping: The animals were randomly selected, 40 animals were equally divided

into five groups of eight animals each. In each group the animals were marked with

numbers to permit individual identification. Each group of 8 animals was kept in separate

polypropylene cages, for one week prior to dosing to allow acclimatization to laboratory

conditions.

Fixation and preparation of rat dose: The normal human adult dose of Shuddha Malla

is 1/120-1/30 Ratti, which is equal to 1mg-4mg. This was converted into animal dose

based on Paget and Barner’s surface area ratio which works out to be 0.36mg /kg body

weight.

Vehicle for Administration of Drug: As the drugs are insoluble, the gum acacia 2%

solution (by mixing 2 gms of gum acacia in 100 ml of distilled water) is taken as the

vehicle and the same as control. The 2% gum acacia is calculated for rat dose of 5 ml

/kg body weight.

90

Procedure: The grouping was done randomly to administer drugs accordingly.

Table No. 17: Shows drugs according to groups.

Group Number of Rats Drug Purpose Group I 8 2% gum acacia soln. To serve as control

Group II 8

Ashuddha Malla

single dose only on 1st

day and 14 days

observation.

To serve as test trial

group.

Group III 8

Ashuddha Malla

single dose for all 14

days.

To serve as test trial

group.

Group IV 8

Shuddha Malla single

dose only on 1st day

14 days observation.

To serve as test trial

group.

Group V 8 Shuddha Malla single

dose for all 14 days.

To serve as test trial

group.

Table 18 : Shows the weight and administered dose for 28 days.

Group Weight of the Rat Calculation of dose Administered dose

Group I ±175 5 x 175 1000

0.75 ml

Group II ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group III ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group IV ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Group V ±175 0.36 x 175 x 100 ml 1000 10 mg

0.63 ml

Administration of Drugs: Drug was administered through intra gastric tube using 2 ml

syringe fitted with 18 gauze needle made of steel provided with 5 number infant feeding

tube to avoid injury to the rats during drug administration. Prescribed dose of suspended

drug was loaded in syringe and the tube was inserted into the oesophagus. After

confirming that the tube was inside the oesophagus, drug was pushed slowly to reach the

gastrum.

Main Study Procedure:

91

� The animals from all groups were administered their respective drug dosage

everyday (1-28 days) as shown in the above tables.

� Body weight was recorded on 0, 3rd, 6th ,9th ,12th ,15th ,18th ,21st ,24th ,27th days of

all the groups to rule out any significant changes.

� Observations: General clinical observations were made at three times a day and

considering the peak period of anticipated effect after dosing. The health

condition of the all the animals was recorded. Thrice daily, all animals were

observed for morbidity and mortality.

� Two animals selected randomly from all the groups were sacrificed on 7th, 14th

,21st and 28th day to trace out the organ toxicity in specific.

Collection of blood samples:- The rats were anaesthetized with Diethyl ether and blood

was collected from retro orbital plexus. The blood samples were collected in Disodium

ethylene diamine tetra acetic acid (EDTA) vials for estimation of haematological

parameters. Serum samples were separated by centrifugation at 5000 rpm for 20 min, and

then were stored at - 80°C until analysis for serum biochemical measurements.

1. Haematology:-The following haematological parameters were determined using blood

samples collected on day 0,7th 14th ,21st and 28th day using microhaematocrit capillary

tubes.

6. Haemoglobin levels(Hb)

7. Total Leucocyte Count (TC)

8. Diffrential Count.(DC)

9. Packed Cell Volume.(PCV)

10. Red Blood Corpuscles (RBC)

2. Serum biochemical parameters:-Serum biochemical parameters were estimated

from the serum samples collected from the animals on Day 0,7and 14th day using a semi

automatic auto analyzer and ready to use kits (Swemed diagnostics, Bangalore).The

following humoral parameters were estimated :-

5. SGOT (Serum glutamic oxalacetate transaminase)/AST

6. SGPT (Serum glutamic pyruvate transaminase)/ALT

7. Total albumin level.

8. Total Protein level.

9. Serum Creatinine.

10. Blood Urea Nitrogen.

92

Histopathological Study:-

On 7th, 14th, 21st and 28th day two animals from all the five groups are sacrificed

to know the significant target organ toxicity, after overnight fasting, the rats were

sacrificed under ether anesthesia. Gross lesions if any were observed.

The main organs like Heart,Brain,Liver,Kidney,Lungs,Spleen and Gastric mucosa

were cleared off from the adnexal tissues using normal saline and placed on a blotting

paper and gently pressed to remove excess saline adhering to the organ. The

representative samples from the organs like liver and kidney were collected in 10%

neutral buffered formalin (NBF) for histopathological study.

The fixed samples were then done for tissue processing,cutting sections and

staining it as described in the previous section. The slides were viewed under a

microscope at various magnifications to note down the microscopic features.

STATISTICAL ANALYSIS: -

The data generated from the experimental study was subjected to one- way

ANOVA by statistical analysis.

93

OBSERVATIONS AND RESULTS Pharmaceutical :- 50 gm of Malla was taken in each of 2 different pots.

� After Shodhana (Swedana) of 50gm of Malla in Karavellaka Swarasa, 46gm and 47

gm of Shodhita Malla was obtained in 1st and 2nd pot respectively.

Analytical:-

♦ Physico-chemical Analysis ,Qualitative and Quantitative results:-

Table 19 :Shows the Physico-chemical results of Samples

Physical tests Ashuddha Malla Shuddha Malla PH Value 6.98 7.08 Ash Value 8.92 % 8.86 % Acid insoluble ash 0.77 % 0.48 % Water soluble ash 0.04 % 0.05 % Loss on drying 0.09 % 0.12 % Loss on ignition 82.84 % 81.39 %

Table No.20: Shows Arsenic (As%) of 2 Samples of Malla.

Quantitative Analysis

Ashuddha Malla Shuddha Malla

As% 69.72% 74.22%

♦X – ray diffraction study of 2 samples of Malla revealed that the composition of

both the samples of Malla has As2O3 (Arsenolite) after comparing the d-Standard

peak values in two different standards of JCPDS.

♦The Particle Size analysis by Laser Diffraction technique shows the size of

Ashuddha Malla as 23.67µm and Shuddha Malla as 43.32µm. .

94

Experimental Results : ACUTE TOXICITY STUDY

STUDY DESIGN:-There are 7 groups each with 8 animals.

� Group 1:Control (gum acacia 2 % solution)

� Group 2:Single dose of Shuddha Malla only on 1st day and 14 days observation.

� Group 3:Single dose of Shuddha Malla on all 14 days .

� Group 4:Dose escalation from 1st day -14th day

� Group 5:Single dose of Ashuddha Malla only on 1st day and 14 days observation.

� Group 6:Single dose of Ashuddha Malla on all 14 days.

� Group 7:Dose escalation from 1st day- 14th day.

OBSERVATIONS:

Behavioural Profile:-

� Group IV and VII are very alert compared to others initially but in later days are

very passive and stereotype.Group III and VI show improved alertness as days

pass.

� The rats from Group IV and VII are restless during initial days.

� Immediate touch response is observed with a spontaneous activity in Groups IV

and VII in the initial days.The same sort of expression is seen in groups III and VI

after 9 days.

Neurological Profile:-

� Convulsions were seen in rats by the 6th-7th hour of high dose administration as

per escalated doses which would die shortly or have an impending death.

� Staggering gait was seen in rats of Group IV and VII in later days.

� There seemed to be some resistance to Paw extension after 3-4 hrs of dosing in

Group IV and VII.

� The Pinna reflex was normal.

Autonomic Profile:-

� Watery Salivation seen in Group IV and VII by 8th and 6th days respectively.

� Diarrhoea was seen with a fluid discharge from the orifice continuously in group

IV and VII by 10th and 8th days respectively.

General Signs:-

� Writhing was noted by most of rats after a particular dosage in escalation from

group IV and VII.

� Reduced food intake in group IV and VII around 7th to 8th day.

95

� Retreat to the corner of the cage or excessive struggling or vocalising on dosing is

seen in group IV and VII as days pass on.

RESULTS OF BODY WEIGHT :-

Table 21: Comparison of body weight between groups.

Group I Group II Group III Group IV Group V Group VI Group VII B. weight

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

0th day

186.38 9.80 183.25 8.50 171.63 7.82 161.63 3.25 161.88 2.36 154.13 3.04 151.25 5.42 <0.001**

3rd day

186.63 9.96 179.88 9.55 171.50 7.67 164.88 5.44 167.38 9.93 156.00 4.63 150.00 7.60 <0.001**

6th day

193.00 12.94 186.13 9.89 178.00 9.70 168.75 5.20 168.00 5.55 158.50 5.50 151.50 9.72 <0.01*

9th day

194.75 12.89 189.13 10.36 180.75 13.61 167.13 6.66 171.25 7.27 165.38 4.31 142.25 14.84 <0.001**

12t

h day

198.00 12.42 188.38 10.18 188.13 13.68 168.40 4.77 168.88 6.38 161.88 5.72 - - <0.01*

14t

h day

195.88 11.63 190.50 11.67 194.38 12.97 169.00 .- 177.50 6.91 167.88 6.27 - - <0.001**

% Change from 0th day

day 3

+0.13 -1.84 -0.07 -2.01 -3.40 -1.22 -0.83 -

day 6 +3.55 +2.57 -3.71 -4.41 +3.78 -2.84 +0.17 -

day 9

+4.49 +3.21 +5.32 -3.40 +5.79 -7.30 -5.95

day 12 +6.24 +2.80 +9.61 +4.19 +4.32 +5.03 -

day 14

+7.10 +3.96 +13.26 +4.56 +9.65 +8.92 -

Table 22:Shows the inter group differences and its significance.

P value Gr.II Gr.III Gr.IV Gr.V Gr.VI Gr.VII

Gr.I 0.688 <0.08+ <0.01** 0.745 0.09+ <0.01**

Gr.II - <0.04* <0.01** 0.093+ <0.05* <0.01**

Gr.III - - <0.03* 0.736 0.812 <0.01**

Gr.IV - - - <0.01** <0.05* 0.589

Gr.V - - - - <0.07+ <0.01**

Gr.VI - - - - - <0.04*

Gr.VII - - - - - -

96

� Highly significant decrease are seen in Group IV and VII when compared to others.

Graph: 6 Shows the body weight in groups

-10

-5

0

5

10

15

% change

Grp I GrpII

GrpIII

GrpIV

Grp V GrpVI

GrpVII

Groups

Body weight Chart

3rd day

6th day9th day

12th day14th day

� Apparent body weight gain is seen in control group.

� By 3rd day all the Groups II,III,IV,and V have a considerable loss of weight.

� Group II and V have a negligible increase by 6th day.

� Group III and VI have a considerable decrease in % of weight.

� Suggestive significant decrease is seen groups IV ,VI and VII by 9th day.

� All the groups show a significant increase by 12th -14th day except group VII as

they were all dead by these days.

2. MORTALITY OF THE RATS RECORDED :-

In groups I,II,III,V, &VI no mortality was observed until the last day (14th

day).All these animals were then sacrificed for collecting organ tissues on 14th day. The

remaining two groups of escalated doses i.e group IV and VII had the death of rats on

particular days with particular doses as shown below in the tables.

Table 23: Showing the number and % of rats dead in the given doses in

Group IV (Shuddha Malla).

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4

97

No.of

Rats

Dead

0/8

0/8

0/8

0/8

0/8

0/8

0/8

0/8

2/8

3/8

7/8

7/8

7/8

7/8

% of

death

0 0 0 0 0 0 0 0 25 37.5 87.5 87.5 87.5 87.5

Table 24: Showing the number and % of rats dead in the given doses in

group VII (Ashuddha Malla)

Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4

No.of

Rats

Dead

0/8

0/8

0/8

0/8

0/8

0/8

1/8

3/8

8/8

-----

-----

-----

-----

-----

% of

death

0 0 0 0 0 0 12.5 37.5 100 ----- ----- ----- ----- -----

Graph 7: Schematic representation of the Quantal dose response curve

98

Graph showing % death due to Shuddha Malla in ranging doses

0 0 0 0 0 0 0 0

25

37.5

87.5 87.5 87.587.5

0102030405060708090

100

0.36 1.8 3.6 5.4 7.2 9 10.8 12.6 14.4 16.2 18 19.8 21.6 23.4

Dose mg/kg body weight

% o

f dea

th o

f rat

s

Foot note:The red line shows the LD 50 and its dose margin.

By the graphical method of Miller and Tainter190:-

The dose when 3 rats were dead was:: 16.2 mg

The dose when 7 rats were dead was:: 18.0 mg

Therefore the dose for 4 rats i.e 50% of the rats to die is :-

18.0-16.2=1.8 mg

When the next 4 rats die with an extra dose of 1.8 mg ::1.8÷4= 0.45

for a single rat.

For 4 rats i.e 50% rats the corresponding probit is-16.2+0.45= 16.65mg is the

predictable LD50. If median efficient dose is taken as 5.4 mg as per dose-response curve

then: Therapeutic index:- LD50/ED50 16.65÷5.4= 3.08.

Graph 8: Schematic representation of the Quantal dose response

curve.

99

Graph showing % of death due to Ashuddha Malla in ranging doses

0 0 0 0 0 012.5

37.5

100

0102030405060708090

100110

0.36 1.8 3.6 5.4 7.2 9 10.8 12.6 14.4 16.2

Dose mg/kg Body weight

% o

f Dea

th o

f rat

s

Foot note: The red line shows the LD 50 and its dose margin.

By the graphical method of Miller and Tainter190:-

The dose when 3 rats were dead was:: 12.6 mg

The dose when 8 rats were dead was:: 14.4mg

Therefore the dose for 4 rats i.e 50% of the rats to die is :-

14.4-12.6=1.8 mg

When the next 5 rats die with an extra dose of 1.8 mg ::1.8÷5= 0.36

for a single rat.

For 4 rats i.e 50% of rats the corresponding probit is -12.6+0.36= 12.96mg is the

predictable LD50.

If median efficient dose is taken as 4.5 mg as per dose-response then:

Therapeutic index:- LD50/ED50 12.96÷4.5= 2.88.

STATISTICAL RESULTS OF BLOOD & SERUM PARAMETERS :-

Table 25: Comparison of Hb between groups.

100

Group I Group II Group III Group IV Group V Group VI Group VII

Hb

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

16.10 0.7 14.75 0.9 14.73 0.6 15.30 1.0 14.83 0.9 15.13 1.0 15.75 0.9 0.015*

7th day

15.14 1.1 13.49 1.4 14.04 1.6 15.69 1.3 14.33 1.1 14.20 1.2 14.91 1.6 0.037*

14th day

13.35 1.7 11.74 0.9 15.61 1.3 16.50 15.05 0.8 15.95 1.5 - - <0.001**

%Change from 1st day

At 7th day

+6.0 -8.6 -4.7 +2.5 -3.4 -6.1 -5.3 -

At 14th day

+17.1 -20.4 +6.0 +7.8 -1.5 +5.5 - -

P value

F=17.501 P=<0.001

F=26.712 P=<0.001

F=6.957 P=0.008

- F=1.776 P=0.205

F=3.982 P=0.043

t=1.326 P=0.226

-

� Group I shows a marginal increase by 7th day and a significant inrease in Hb% by

14th day.

� The Group II shows negligible decrease from 1st -7th day and then from 7th -14th

day a suggestive significant decrease.

� In group III there is negligible decrease from 1st-7th day,there is a negligible

increase in % change from 7th-14th day.

� In group IV there is negligible increase in Hb% by 14 days.

� In group V there is a negligible decrease from 1st-14th day.

� In group VI though there is negligible decrease in 7 days then a negligible

increase by 14th day.

� In group VI I there is negligible decreasing of Hb% from 1st -7th day.

Table 26:Comparison of TLC between groups.

Group I Group II Group III Group IV Group V Group VI Group VII

Total count

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

11600 961 10525 843 11600 711 11468 1037 10837 1225 11312 1135 11500 1177 0.262

101

7th day

10412 1129 10118 1005 10718 980 10450 892 10631 1057 10018 815 10437 1067 0.795

14th day

10237 1315 9818 939 10575 1045 9500 10843 707 10237 582.9 - - 0.325

%Change from 1st day

At 7th day

-10.2 -3.9 -7.6 +58.9 -1.9 -11.4 +79.2 -

At 14th day

-11.7 +6.7 -8.8 +17.2 +0.1 -9.5 - -

P value

F=16.066 P=<0.001

F=4.699 P=0.058

F=6.344 P=0.011

- F=0.296 P=0.748

F=8.042 P=0.005

t=2.653 P=0.033

-

� In Group I TLC is negligibly reduced by 14th day.

� In group II TLC % is negligibly decreased and increased by 14th day.

� In group III TLC % is negligibly decreased.

� In group IV TLC% is significant increase by 7 days and by 14th day it is increased

significantly.

� In group V the % of TLC has a negligible decrease.

� In group VI the TLC % has decreased negligibly.

� In group VII there is highly significant increase in 7 days.

Table 27: Comparison of Differential Counts-Polymorphs between

groups.

Group I Group II Group III Group IV Group V Group VI Group VII

DC-P

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 19.38 5.3 23.13 2.2 23.63 4.5 24.50 3.0 23.25 2.2 24.13 4.9 23.13 2.7 0.161

7th day 26.00 4.2 24.38 3.8 23.50 4.8 26.75 3.6 23.00 3.9 24.13 2.2 25.50 4.2 0.435

14th day 27.38 4.4 22.63 2.7 24.38 3.1 26.00 25.00 3.7 28.75 5.6 - - 0.062+

%Change from 1st day

102

At 7th day

+34.2 5.4 -0.5 9.2 -1.1 0.0 10.3 -

At 14th day

+41.3 -2.2 3.2 6.1 7.5 19.2 - -

P value F=9.793 P=0.002

F=0.919 P=0.422

F=0.131 P=0.878

- F=1.047 P=0.377

F=3.434 P=0.061

t=1.239 P=0.255

-

� In group I there is moderately significant increase by 7th and by 14th day.

� In group II there is negligible increase in 7 days but later a negligible decrease.

� In group III there is no change but later there is a negligible increase.

� In group IV there is a negligible increase by 7th and 14th day..

� In group V there is no change in 7 days but by 14th day there is a negligible

increase.

� In group VI there is no change absolutely but there a significant increase by 14th

day.

� In group VII there is a negligible increase in 7 days.

Table 28:Comparison of Lymphocytes between groups.

Group I Group II Group III Group IV Group V Group VI Group VII DC-

L Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

81.38 5.7 73.88 1.9 77.63 5.3 76.25 5.4 74.13 2.5 77.38 6.4 76.00 4.2 0.052+

7th day

69.63 5.0 71.00 4.1 73.38 4.1 70.50 4.3 72.13 3.4 72.00 1.3 71.38 4.6 0.616

14th day

71.00 2.4 68.75 5.8 72.75 2.0 80.00 71.00 4.2 76.13 7.9 0.050+

%Change from 1st day

At 7th day

-1.4 -3.9 -5.5 -7.5 -2.7 -6.9 -6.1 -

At 14th day

-1.7 -6.9 -6.3 14.9 -4.2 -11.6 - -

P value

F=13.684 P=0.001

F=4.807 P=0.026

F=3.926 P=0.044

- F=2.264 P=0.141

F=1.949 P=0.179

t=2.499 P=0.041

-

� In group I , II & III there is negligible decrease in lymphocyte % from 1st to 14th

day.

103

� In group IV negligible decrease by 7 days then later 7 days the % change has

increased significantly.

� In group V the DC-L% has decreased slightly from 1st to 14th day.

� In group VI the % of DC-L has decreased negligibly.

� In group VII the % change of DC-L is negligibly decreased in 7 days.

Table 29:Comparison of Eosinophils between groups.

Group I Group II Group III Group IV Group V Group VI Group VII DC-

E Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

1.50 1.2 1.13 0.8 1.00 0.5 1.00 0.9 0.88 0.6 1.13 0.6 1.13 0.6 0.819

7th day

1.63 1.2 1.88 0.8 1.88 1.4 1.00 0.9 2.50 1.1 2.13 1.2 1.50 1.4 0.257

14th day

1.13 1.1 1.50 0.5 1.00 0.8 2.00 1.38 0.5 1.38 0.5 - - 0.627

%Change from 1st day

At 7th day

+8.3 +66.7 +7.5 0.0 +85.7 +88.9 +33.3 -

At 14th day

-25.0 +33.3 0.0 -80.0 +57.1 +22.2 - -

P value

F=0.532 P=0.001

F=4.807 P=0.026

F=3.926 P=0.044

- F=9.404 P=0.003

F=3.138 P=0.078

t=0.629 P=0.549

-

� In group I there is a negligible increase by 7th day, significant decrease by 14th

day.

� In group II there is a moderately significant increase by 14th day.

� In group III there is a negligible increase by 7 days .

� In group IV there is highly significant increase by 14th day.

� In group V there is highly significant increase which moderately increases by 14th

day.

� In group VI there is highly significant increase by 7th day,a suggestive significant

increase by 14th day.

� In group VII there is moderately significant increase by 7 days.

Table 30:Comparison of Monocytes between groups.

104

Group I Group II Group III Group IV Group V Group VI Group VII

DC-M

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 2.25 1.0 2.38 1.3 2.25 0.9 1.63 0.7 2.00 1.5 2.50 0.9 1.75 0.9 0.619

7th day 2.25 1.0 1.38 1.3 1.38 1.8 1.63 1.2 1.38 1.1 1.38 1.4 0.88 0.6 0.531

14th day

2.25 1.4 1.00 0.8 1.13 1.0 - - 1.50 0.9 2.38 1.9 - - 0.114

%Change from 1st day

At 7th day

0.0 -42.1 -38.9 +5.0 -31.3 -45.0 -50.0 -

At 14th day

0.0 -57.9 +50.0 - -25.0 -5.0 - -

P value

F=0.000 P=1.000

F=3.158 P=0.074

F=2.122 P=0.157

- F=0.605 P=0.560

F=1.231 P=0.332

t=2.966 P=0.021

-

� In group I there is no change absolutely.

� In group II there is moderately significant decrease from 1st-14th day.

� In group III there is moderately significant decrease by 7th day ,a moderately

significant increase by 14th day.

� In group IV there is negligible increase by 7 days.

� In group V there is significant decrease by 14th day.

� In group VI there is moderately significant decrease by 7th day.

� In group VII there is moderately significant decrease by 7th day.

Table 31:Comparison of Packed Cell Volume between groups.

Group I Group II Group III Group IV Group V Group VI Group VII PCV

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

49.71 3.9 45.78 2.3 47.10 5.0 49.43 6.4 48.03 5.4 48.26 5.9 50.84 4.4 0.465

7th day

44.41 5.9 41.86 4.3 43.44 4.0 43.63 4.7 42.98 3.3 42.65 3.6 45.03 4.2 0.823

14th day

41.73 5.9 39.45 4.6 43.76 5.6 49.20 44.80 3.6 43.71 3.6 - - 0.175

%Change from 1st day

105

At 7th day

+10.7 -8.5 -7.8 -11.7 -10.5 -15.6 -11.4 -

At 14th day

+16.1 -13.8 +7.1 -0.5 -6.7 -9.4 - -

P value

F=11.557 P=0.001

F=3.544 P=0.001

F=4.948 P=0.024

- F=2.520 P=0.116

F=6.261 P=0.011

t=3.077 P=0.018

-

� In group I has increased significantly by14th day.

� In group II % has decreased negligibly.

� In group III negligible decrease by 7th day and a negligible increase by 14th day.

� In group IV there is a significant decrease in 7 days.

� In group V there is a negligible decrease by 14th day.

� In group VI there is significant decrease by 7th day.

� In group VII a significant decrease is seen by 7 days.

Table 32:Comparison of Red blood Corpuscles between groups.

Group I Group II Group III Group IV Group V Group VI Group VII RBC

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

5.93 0.3 5.44 0.5 5.39 0.5 6.01 0.6 5.41 0.6 5.61 0.9 5.97 0.9 0.204

7th day

4.92 0.5 4.77 0.7 4.86 0.6 5.04 0.4 4.77 0.4 4.73 0.4 5.14 0.5 0.581

14th day

4.55 0.7 4.13 0.6 5.95 1.0 5.38 5.53 0.5 6.31 1.1 - - <0.001**

%Change from 1st day

At 7th day

+16.9 +12.4 -9.9 -16.0 -11.9 -15.7 -13.8 -

At 14th day

+23.3 -4.1 10.4 -10.4 2.1 -12.4 - -

P value

F=29.437 P<0.001

F=16.829 P=0.001

F=6.105 P=0.002

- F=5.875 P=0.014

F=5.210 P=0.050

t=1.999 P=0.086

-

� In group I there is significant increase by14th day.

� In group II though there is significant increase by 7 days.

106

� In group III there is negligible decrease by 7th day, and a negligible increase by

14th day.

� In group IV there is significant decrease by 7th day.

� In group V there is suggestive significant decrease by 7th day .

� In group VI there is significant decrease from 1st-14th day.

� In group VII there is significant decrease in 7 days.

Table 33:Comparison of Urea between groups.

Group I Group II Group III Group IV Group V Group VI Group VII P value

Urea

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

1st day

29.13 2.1 31.81 4.5 40.36 5.6 29.41 2.0 31.59 4.1 36.04 8.1 33.09 5.6 0.001**

7th day

39.78 9.5 30.44 5.4 42.85 11.6 55.40 11.5 34.15 5.5 59.26 17.9 56.49 13.4 <0.001**

14th day

39.73 10.6 34.01 3.7 45.23 11.8 69.90 33.40 5.9 57.05 19.5 - - 0.001**

%Change from 1st day

At 7th day

36.5 -4.3 6.2 88.4 8.1 64.4 70.7 -

At 14th day

36.4 6.9 12.0 97.7 5.7 58.3 - -

P value

F=4.063 P=0.045

F=1.425 P=0.273

F=1.497 P=0.261

- F=0.520 P=0.606

F=8.710 P=0.003

t=5.285 P=0.001

-

� In group I there is moderately significant increase by 14th day.

� In group II it has decreased negligibly by 7 days.

� In group III negligible increase by 14th day.

� In group IV highly significant increase by 7th , 14th day.

� In group V the negligible increase by 14th day.

� In group VI increased moderately by14th day.

� In group VII there is highly significant increase by 7th day.

107

Table 34:Comparison of Creatinine between groups.

Group I Group II Group III Group IV Group V Group VI Group VII P

value Creatinine

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

1st day 0.87 0.3 1.00 0.4 1.03 0.3 1.35 0.4 0.67 0.1 0.91 0.5 0.77 0.4 0.011*

7th day 0.73 0.2 0.87 0.3 0.66 0.2 0.72 0.1 0.66 0.1 0.66 0.2 0.72 0.2 0.371

14th day 0.69 0.2 0.59 0.1 0.77 0.2 0.82 0.68 0.1 0.76 0.3 - - 0.473

%Change from 1st day

At 7th day -1.3 12.9 35.2 46.7 1.9 26.9 6.3 -

At 14th day

-2.6 40.5 25.2 39.1 2.2 16.4 -

P value F=0.868 P=0.445

F=6.487 P=0.010

F=6.144 P=0.012

- F=0.104 P=0.902

F=2.180 P=0.150

t=0.388 P=0.709

-

� In group I there is a negligible decrease by 14 days.

� In group II there is moderate increase in 7 days but a significant increase in 14

days.

� In group III there is moderately significant increase seen by 14th day.

� In group IV there is moderately significant increase by 14 days.

� In group V there is negligible increase by 14th day.

� In group VI there is moderately significant increase by 14th day.

� In group VII there is negligible increase by 7 days.

Table 35 :Comparison of SGOT between groups

Group I Group II Group III Group IV Group V Group VI Group VII

SGOT

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 51.50 8.6 63.50 9.2 59.13 10.2 61.88 8.5 48.50 8.8 56.25 10.3 52.75 8.6 0.016*

7th day 51.63 5.3 67.75 18.8 49.63 6.8 83.50 6.8 48.50 3.7 67.75 16.8 86.63 11.3 <0.001**

14th day

53.88 7.5 53.50 11.9 54.63 6.5 86.00 54.13 5.3 73.00 20.0 - - 0.002**

%Change from 1st day

At 7th day

0.2 +6.7 +6.1 +14.9 +9.0 +20.4 +64.2 -

At 14th day

4.6 +5.7 +7.6 +19.0 +11.6 +29.8 - -

108

P value

F=0.278 P=0.761

F=2.747 P=0.099

F=6.250 P=0.034

- F=3.115 P=0.076

F=8.041 P=0.005

t=7.725 P<0.0001

-

� In group I negligible increase is seen.

� In group II there is negligible increase by 14th day.

� In group III there is negligible increase by 14th day

� In group IV there is suggestive significant increase in % by 14th day.

� In group V there is suggestive significant increase by 14th day.

� In group VI there is suggestive significant increase seen by 14th day.

� In group VII there is moderately significant % change increased by 7th day.

Table 36:Comparison of SGPT between groups.

Group I Group II Group III Group IV Group V Group VI Group VII

SGPT

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 155.25 13.1 167.88 8.4 163.50 8.5 173.88 11.6 177.13 21.2 170.75 4.2 170.13 8.0 0.015*

7th day 163.63 25.0 201.13 9.8 164.75 6.9 186.75 3.6 209.00 19.8 192.25 25.9 187.25 17.8 <0.001**

14th day

165.88 24.5 171.50 17.7 171.75 8.7 24.13 68.2 217.50 34.0 195.13 33.1 - - <0.001**

%Change from 1st day

At 7th day

+0.4 +9.8 +0.8 +7.4 +18.0 +12.6 +30.1 -

At 14th day

+1.8 +2.2 +5.0 +86.1 +22.8 +14.3 - -

P value F=1.677 P=0.222

F=19.888 P<0.001

F=2.066 P=0.164

- F=6.889 P=0.025

F=4.951 P=0.024

t=2.124 P=0.071

-

� In group I there is a negligible increase.

� In group II there is negligible increase.

� In group III there is negligible increase.

� In group IV there is negligible increase by 7th day,a highly significant increase by

14th day.

� In group V moderately significant change seen in 14 days.

� In group VI moderate increase is observed for 14 days.

� In group VII highly significant increase in is obtained.

109

Table 37:Shows the HISTOPATHOLOGY RESULTS:-

G

R

O

U

P

HEART BRAIN LIVER KIDNEY LUNGS SPLEEN GASTR

-IC

MUCO

—SA

I Normal Normal Normal Normal Mild

Congestion

Oedema

Normal Normal

II Normal Normal Normal Normal ---same----- Normal Normal

II

I

Normal Normal Normal Normal -----same----- Normal Mild

Lesion

I

V

Destruction

Of cardiac

Fibres.

Mild

Congestion,

Degenerating

Neurons.

Mild

fatty

infiltrations

severe

haemorrhage.

Mild

congestion

Degeneration,

Oedema,

Haemorrhage

Infiltration of

inflammatory

cells in

interstitial

Mild

congestion Mild

keratiniz-

ation,desc

-umation

of krypts

V Normal Normal Normal Normal Mild congn Normal Normal

V

I

Congestion

Of cells.

Mild lesion ,

haemorrhage Congestion

Of cells

Periportal

infiltration

Atrophic

Glomeruli,

Tubular

Changes,

necrosis

Severe

haemorrhage

around the

vessel,thicken

-ing of

alveolar wall.

Normal Villi

showing

descumat

-ion

,destructi

-on

V

II

Severe

haemorrhag

-e &

destruction

of muscle

fibres.

Congestion

Mild swollen

oligodendrites

Infiltration of

Lymphocytes.

Destruction

Of

hepatocytes

Remnants

Of

Epithelium.

Congestion,

Haemorrhage

Necrosis of

Tubular

epithelium

with

proteinacious

casts,swollen

tubules.

Periartiritis,

Thickening of

alveolar

wall,haemorr

hage

Congestion

haemorrha

ge,prolifera

tion of

lymphoblas

ts around

the

arteriole.

Descumat

-ion due

to

irritation.

110

SUB-ACUTE STUDY STUDY DESIGN: Single dose and repeated dose toxicity study within 5 groups each

with 8 animals is undertaken to study the toxicity based on different parameters..

� Group 1: Control

� Group 2: Single therapeutic dose of Shuddha Malla only on 1st day and 28 days

observation.

� Group 3: Single therapeutic dose of Shuddha Malla on all 28 days.

� Group 4: Single therapeutic dose of Ashuddha Malla only on 1st day and 28 days

observation.

� Group 5: Single therapeutic dose of Ashuddha Malla on all 28 days.

GENERAL OBSERVATIONS:

� The group 3 and 5 showed an improved alertness compared to other groups.

� Increased motor activity is observed in groups 3 and 5.

� The Pinna reflex was normal in all groups.

� The colour change in faecal matter from straw yellow to coffee brown was

observed by 16th and 18th days in group 5 and 3 respectively.

RESULTS OF THE BODY WEIGHT :

Table 38: Comparison of weight between groups.

Group I Group II Group III Group IV Group V Body weight

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

0th day 153.63 4.57 156.00 4.54 152.63 2.33 156.63 12.41 153.50 2.62 0.679

3rd day 158.50 6.55 157.00 6.87 147.25 3.54 155.75 14.08 157.38 5.21 0.059+

6th day 171.88 8.37 167.13 9.36 153.88 2.36 163.75 16.54 165.88 4.49 0.011*

9th day 172.00 7.72 165.67 9.65 156.00 3.46 166.50 20.58 161.50 3.89 0.166

12th day 181.50 7.99 179.17 14.48 171.17 3.19 185.67 22.50 176.00 5.93 0.388

15th day 184.50 12.45 187.50 17.82 175.75 7.89 182.75 2.87 183.50 3.70 0.20

18th day 202.00 18.17 202.25 18.45 187.75 13.15 198.75 2.75 200.00 5.16 0.533

21st day 213.25 25.25 204.50 18.08 202.75 13.57 201.00 13.22 206.50 4.80 0.851

24th day 240.50 45.96 207.00 26.87 212.00 0.00 214.50 7.78 208.50 2.12 0.651

111

27th day 216.50 40.31 214.00 28.28 214.00 11.31 218.50 26.16 217.00 7.07 1.000

% Change from 0th day

3rd day 3.17 -0.64 -3.52 -0.56 -2.52 -

6th day 11.88 7.13 -0.82 4.55 -8.06 -

9th day 11.96 6.20 2.21 6.30 -5.21 -

12th day 18.14 14.85 12.15 18.54 14.66 -

15th day 20.10 20.19 15.15 16.68 19.54 -

18th day 31.49 29.65 23.01 26.90 30.29 -

21st day 38.81 31.09 32.84 28.33 34.53 -

24th day 56.55 32.69 38.90 36.95 35.83 -

27th day 40.93 37.18 40.21 39.51 41.37 -

Graph 9: Schematic representation of differences in body weight.

Body weight Chart

-20-10

0102030405060

Grp I Grp II Grp III Grp IV Grp V

Groups

% C

han

ge

3rd day

6th day

9th day

12th day

15th day

18th day

21st day

24th day

27th day

� Normal body weight gain is seen in Group I.

� On 3rd day there is a considerable suggestively significant decreased percentage of

weight loss in Groups II, III,IV and V.

� Group II when compared to III, IV and V has a significant increase in body

weight by 6th day.

� Group III the significant increase is seen only by 9th day.

� Group IV also shows significant increase from 6th day but to lesser percentage

compared to Group II.

� Group V has suggestive significant decrease until 9th day then by 12th day a

significant increase is shown.

112

STATISTICAL RESULTS OF BLOOD & SERUM PARAMETERS

Table 39: Comparison of Hb between groups.

Group I Group II Group III Group IV Group V

Hb

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 15.1 0.6 16.5 1.2 16.7 0.4 16.7 0.4 16.4 0.8 0.001**

7th day 13.9 0.4 15.7 1.2 15.7 0.4 15.6 0.4 15.5 0.7 <0.001**

14th day 12.9 1.2 13.4 0.9 20.8 1.3 14.9 1.0 13.0 1.2 0.009**

21st day 14.2 0.5 14.3 0.6 24.6 0.7 14.7 0.2 13.6 0.2 0.041*

28th day 14.8 0.4 14.0 0.1 13.9 1.4 14.4 0.3 23.7 1.1 0.480

% Change from 1st day

At 7th day -8.2 -4.5 -6.2 -6.6 -5.0 -

At 14th day -14.9 -18.6 +7.2 -10.4 -20.9 -

At 21st day -6.5 -13.1 +12.9 -11.6 -16.9 -

At 28th day -2.6 -15.2 +7.1 -13.5 +16.3 -

� In group I there is suggestive decrease in % of Hb.

� In group II also a suggestive significant decrease is seen from 7th -28th day.

� In group III a moderate increase is seen by 21st day.

� In group IV there is a significant decrease from 7th-28th day.

� In group V there is significant Hb% decrease until 21st day but later by 28th day

there is a significant increase.

Table 40: Comparison of Total count between groups.

Group I Group II Group III Group IV Group V Total count

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day

8987.5 1035.7 10737.5 1536.2 10425.0 554.8 11325.0 660.6 10587.5 653.4 <0.001**

7th day

8125.0 1203.3 10012.5 1652.2 10168.8 459.0 10525.0 406.2 10156.3 550.0 <0.001**

14th day

8750.0 1385.3 9041.7 1774.9 10600.0 720.4 10383.3 1162.2 8200.0 1714.4 0.025*

21st day

12900.0 2891.4 10387.5 611.5 9850.0 - 10600.0 679.5 9150.0 669.6 0.056+

28th day

8500.0 141.4 9075.0 813.2 7550.0 919.2 10550.0 989.9 10550.0 989.9 0.055+

113

% Change from 1st day

At 7th day

-9.6 -6.8 -2.5 -7.1 -4.1 -

At 14th day

-2.6 -15.8 +1.7 -8.3 -22.6 -

At 21st day

+43.5 -3.3 -5.5 -6.4 -13.6 -

At 28th day

-5.4 -15.5 -27.6 -6.8 -0.4 -

� In group I there is moderate decrease by 14th day but a moderate significant %

increase is seen by 21st day and again a slight decrease by 28th day.

� In group II there is a moderately significant decrease from 7th to 28th day.

� In group III though by 7th day there is marginal decrease and a moderate

decrease by 28th day.

� In group IV a negligible decrease is seen.

� In group V significant decrease is observed by 14th and 21st day.

Table 41: Comparison of DC-P between groups.

Group I Group II Group III Group IV Group V

DC-P

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 51.1 4.4 57.0 6.0 58.5 4.3 58.4 3.2 53.9 5.6 0.016*

7th day 48.3 5.4 53.4 6.1 49.5 5.7 50.0 8.1 53.4 1.7 0.279

14th day 47.3 3.6 52.2 5.5 51.5 4.9 54.3 4.4 49.8 6.4 0.201

21st day 43.0 4.4 49.0 7.4 52.8 6.4 45.8 9.6 39.0 2.9 0.078+

28th day 47.5 7.8 53.0 1.4 46.5 2.1 50.0 14.1 55.5 0.7 0.722

% Change from 1st day

At 7th day -5.6 -6.4 -15.4 -14.3 -0.9 -

At 14th day -7.4 -8.5 -12.0 -6.9 -7.5 -

At 21st day -15.9 -14.0 -9.8 -21.6 -27.6 -

At 28th day -7.1 -7.0 -20.5 -14.3 +3.0 -

� In group I & II there is negligible change in polymorphs % but significant by

21st day.

114

� In group III significance decrease is seen by 28th day.

� In group IV decrease is significant by 21st day.

� In group V the decrease is significant by 21st day but by 28th day there is a

negligible increase.

Table 42: Comparison of DC-L between groups.

Group I Group II Group III Group IV Group V

DC-L

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 56.4 3.2 46.9 7.2 77.6 5.3 52.8 2.6 48.4 2.6 <0.001**

7th day 52.0 3.0 49.3 6.1 48.8 6.7 48.3 6.4 43.3 2.5 0.035*

14th day 48.8 4.2 46.0 7.6 47.7 5.3 45.3 5.0 45.0 7.8 0.790

21st day 55.5 3.5 49.5 5.8 45.3 7.1 52.0 10.0 60.8 3.0 0.037*

28th day 53.0 9.9 46.0 2.8 46.5 6.4 46.0 14.1 43.0 1.4 0.809

% Change from 1st day

At 7th day -7.8 +5.1 -37.2 -8.5 -10.6 -

At 14th day -13.4 -1.9 -38.6 -14.1 -7.0 -

At 21st day -1.6 +5.6 -41.7 -1.4 +25.6 -

At 28th day -6.0 -1.9 -40.1 -12.8 -11.1 -

� In group I there is a negligible decrease in lymphocyte %.

� In group II there is negligible increase and later by 21st day there is a negligible

increase.

� In group III there is a moderately significant decrease by 7th ,14th,21st and 28th

days.

� In group IV there is a negligible decrease by 7th day,a significant decrease by 14th

and 28th day.

� In group V negligible decrease is seen by 14th day but later by 21st day a

significant increase is seen.

Table 43: Comparison of DC-E between groups.

DC-E Group I Group II Group III Group IV Group V P value

115

Mean SD Mean SD Mean SD Mean SD Mean SD

1st day 2.6 1.5 1.1 0.8 1.0 0.5 2.0 1.3 1.4 0.9 0.025*

7th day 0.9 1.0 1.1 0.6 1.6 0.7 1.1 0.6 1.4 0.9 0.412

14th day 0.5 0.5 1.2 1.2 1.2 1.2 0.7 0.8 1.7 0.5 0.201

21st day 0.5 0.6 1.3 1.5 0.8 1.0 0.8 1.0 1.3 1.0 0.790

28th day 0.5 0.7 0.5 0.7 1.5 2.1 1.0 1.4 0.5 0.7 0.898

% Change from 1st day

At 7th day -66.7 -0.0 +62.5 -43.8 -70.0 -

At 14th day -81.0 +3.7 +16.7 -66.7 +21.2 -

At 21st day -81.0 +11.1 -25.0 -62.5 -9.1 -

At 28th day -81.0 -55.6 +50.0 -50.0 -63.6 -

� In group I a moderately significant decrease is observed on 7th,14th,21st,28th days.

� In group II there is no or negligible change seen by 21st day but a moderately

significant decrease is seen by 28th day.

� In group III there is a moderately significant increase by 7th day but later by 21st

day a significant decrease and a moderate increase by 28th day.

� In group IV there is a moderate decrease on all 7th ,14th,21st and 28 days.

� In group V there is a highly significant decrease by 7th day but by 14th ,21st day a

negligible increase and a moderately significant decrease by 28th day.

Table 44: Comparison of DC-M between groups.

Group I Group II Group III Group IV Group V

DC-M

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 2.6 1.1 2.4 1.3 1.4 0.7 1.3 1.0 1.3 1.0 0.023*

7th day 1.6 0.5 1.8 1.0 2.9 1.6 1.8 0.5 2.4 0.7 0.070+

14th day 2.2 0.8 1.5 0.5 1.2 1.0 1.0 0.9 1.8 0.4 0.073+

21st day 1.3 1.0 0.8 0.5 2.0 0.8 2.0 0.8 1.0 0.8 0.133

28th day 1.5 0.7 1.0 1.4 1.5 0.7 2.0 0.0 1.5 0.7 0.830

% Change from 1st day

116

At 7th day -38.1 -26.3 +100.1 +40.0 +90.0 -

At 14th day -17.5 -36.8 -15.2 -20.0 +46.7 -

At 21st day -52.4 -68.4 +45.5 +60.0 -20.0 -

At 28th day -42.9 -57.9 +9.1 +60.0 +20.0 -

� In group I is moderately % decreased by 7th ,21st,28th days.

� In group II significant decrease by 7th day and moderately significant decrease by

14th,21st and 28th day.

� In group III a highly significant increase is seen in % by 7th day and a negligible

decrease by 14th day,a moderate increase by 21st day,negligible increase by 28th

day.

� In group IV a moderately significant increase is seen by 7th day,21st and 28th day

but a suggestive decrease is found by 14th day.

� In group V there is highly significant % increase by 7th day,moderate increase by

14th,a significant decrease by 21st,a significant increase by 28th day.

Table 45: Comparison of RBC between groups.

Group I Group II Group III Group IV Group V

RBC

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 7.3 1.0 8.7 0.9 9.2 0.3 9.3 0.5 8.7 0.5 <0.001**

7th day 6.0 0.5 6.0 0.5 8.4 0.4 7.9 0.9 7.5 0.7 <0.001**

14th day 5.9 0.8 6.6 1.1 6.6 0.7 6.6 0.7 5.9 0.9 0.350

21st day 6.0 0.8 6.5 0.6 6.8 0.5 6.4 0.6 5.1 0.1 0.005**

28th day 5.3 1.0 6.5 0.7 6.3 0.4 7.0 0.2 40.5 2.1 <0.001**

% Change from 1st day

At 7th day -17.6 -30.4 -9.1 -14.9 -14.2 -

At 14th day -19.6 -23.5 -28.3 -28.6 -31.8 -

At 21st day -18.9 -25.3 -26.0 -30.8 -41.4 -

At 28th day -27.8 -25.4 -31.4 -24.8 -85.9 -

� In group I there is a suggestive significant decrease % of RBC.

� In group II there is a significant decrease in RBC %.

117

� In group III a negligible decrease by 7th day but a significant decrease by

14th,21st,28th day.

� In group IV a significant decrease is seen by 7th,14th,21st and 28th day.

� In group V a moderate decrease is seen by 21st day ,a highly significant decrease

by 28th day.

Table 46: Comparison of PCV between groups.

Group I Group II Group III Group IV Group V

PCV

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 43.7 2.6 48.8 1.6 51.4 2.9 50.7 2.2 49.3 1.5 <0.001**

7th day 41.5 0.6 46.7 1.5 46.6 1.2 46.7 1.4 46.3 2.0 <0.001*

14th day 39.6 5.1 44.0 3.1 40.6 1.1 57.4 9.5 38.3 3.0 <0.001**

21st day 45.1 4.8 46.3 3.6 46.6 5.6 48.4 4.5 40.3 1.7 0.136

28th day 45.0 7.1 42.5 2.1 41.0 1.4 45.5 0.7 40.5 2.1 0.556

% Change from 1st day

At 7th day -5.1 -4.3 -9.4 -7.9 -6.1 -

At 14th day -9.4 -9.9 -21.1 +13.2 -22.2 -

At 21st day +3.2 -5.3 -9.5 -4.5 -18.3 -

At 28th day +2.9 -13.0 -20.3 -10.3 -17.8 -

� In group I a negligible increase and decrease % of PCV.

� In group II a negligible decrease is seen in PCV %.

� In group III a negligible decrease is seen by 7th and 21st day,a significant decrease

by 14th and 28th day.

� In group IV a negligible decrease is seen by 7th ,21st,28th and a significant increase

by 14th day.

� In group V a negligible decrease is seen by 7th day,a significant increase by

14th,21st and 28th day.

Serum analysis:- Table 47: Comparison of SGOT between groups.

SGOT Group I Group II Group III Group IV Group V P value

118

Mean SD Mean SD Mean SD Mean SD Mean SD

1st day 160.0 27.0 165.1 18.5 166.5 20.1 166.7 15.4 167.0 25.0 0.965

7th day 226.9 92.1 211.0 11.5 226.9 92.1 196.5 61.4 200.5 58.0 0.850

14th day 105.8 77.7 159.6 28.3 161.9 19.3 168.1 9.6 105.8 77.7 0.094+

21st day 135.3 33.9 134.9 32.1 182.1 12.0 182.1 12.0 144.9 22.8 0.061+

28th day 136.7 22.7 119.7 12.6 161.2 74.9 113.7 3.7 94.4 6.3 0.485

% Change from 1st day

At 7th day 41.8 27.8 36.3 17.8 20.0 -

At 14th day -33.9 -3.4 -2.8 0.8 +36.6 -

At 21st day -15.4 -18.3 +9.4 9.3 +13.2 -

At 28th day -14.6 +7.5 +.2 -31.8 +43.5 -

� In group I a moderately significant increase is seen by 7th day, then a moderate

decrease by 14th day,a significant decrease by 21st and 28th day.

� In group II a suggestive significant increase is seen by 7th day but a negligible

decrease by 14th day,a suggestive significant decrease by 21st and 28th day.

� In group III a moderately significant increase is seen by 7th day,a negligible

decrease by 14th and 28th day,a negligible increase by 21st day.

� In group IV a suggestive significant increase by 7th day,a negligible decrease by

21st day,a moderately significant decrease by 28th day.

� In group V a suggestive increase in change by 7th day and 21st day,a moderate

significant increase by 14th day and 28th day.

Table 48: Comparison of SGPT between groups.

Group I Group II Group III Group IV Group V

SGPT

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 45.1 5.6 43.0 6.3 49.8 7.4 43.1 9.6 50.8 13.5 0.294

7th day 69.6 2.8 52.0 15.1 69.6 2.8 79.8 21.2 91.7 3.5 <0.001**

14th day 53.5 1.5 56.2 3.7 65.5 3.6 72.3 7.0 73.4 5.0 <0.001**

21st day 42.3 6.6 42.3 6.6 77.5 5.2 73.7 18.0 72.4 4.3 <0.001**

28th day 9.6 12.5 9.6 12.5 5.3 2.5 3.9 12.5 84.6 3.4 0.912

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% Change from 1st day

At 7th day 54.2 20.9 39.9 85.0 80.7 -

At 14th day 18.6 30.7 31.7 67.7 44.6 -

At 21st day -6.3 -1.7 55.8 70.9 42.7 -

At 28th day -78.8 -77.7 69.3 90.9 91.0 -

� In group I a moderate increase in SGPT by 7th day, a suggestive significance by

14th day, a negligible decrease by 21st day,a highly significant decrease in % by

28th day.

� In group II a suggestive significant increase by 7th and 14th day, a highly

significant decrease is seen by 28th day.

� In group III a moderately significant increase by 7th, 14th ,21st,28th day.

� In group IV a highly significant increase % is seen by 7th day,a moderately

significant increase by 14th and 21st day,a highly significant increase by 28th day.

� In group V a highly significant increase by 7th day,a moderate increase by 14th and

21st, a highly significant increase by 28th day.

Table 49: Comparison of Albumin between groups.

Group I Group II Group III Group IV Group V

Albumin

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 4.3 0.8 43.0 6.3 4.8 0.7 48.4 0.8 4.9 0.9 <0.001**

7th day 3.3 1.1 3.3 1.1 6.6 1.0 5.6 1.0 6.4 1.3 <0.001**

14th day 6.3 0.9 5.1 0.9 5.2 0.5 6.0 0.9 6.1 1.3 0.128

21st day 5.6 0.7 3.8 1.9 4.5 0.3 5.1 1.0 3.1 2.3 0.170

28th day 4.8 0.2 4.8 0.2 5.6 0.7 5.5 0.2 3.0 1.3 0.070+

% Change from 1st day

At 7th day -22.2 -92.3 38.1 -96.4 30.4 -

At 14th day 46.5 -88.1 7.5 -94.5 25.5 -

At 21st day 30.6 -91.1 -5.6 -84.3 -36.6 -

At 28th day 11.3 -88.9 15.5 -94.2 -38.3 -

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� In group I a suggestive decrease by 7th day,a moderate increase by 14th day,21st

day, a significant increase by 28th day.

� In group II a highly significant decrease in albumin by 7th,14th,21st,28th days.

� In group III a moderate increase by 7th day,a negligible increase by 14th,a

moderate increase by 28th day.

� In group IV a highly significant decrease is seen by 7th -28th day.

� In group V a suggestive increase by 7th and 14th day,a moderate decrease by 21st

and 28th day.

Table 50: Comparison of TPR between groups.

Group I Group II Group III Group IV Group V

TPR

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 7.9 1.8 8.7 2.6 7.2 1.5 9.0 1.8 9.1 2.9 0.377

7th day 9.4 1.4 12.3 0.8 12.8 1.6 11.5 0.8 8.7 1.0 <0.001**

14th day 10.8 2.6 11.6 1.6 12.6 1.7 13.4 1.6 13.8 1.8 0.065+

21st day 5.9 1.9 6.7 2.3 8.8 5.2 7.1 2.4 - - 0.640

28th day 11.6 0.8 11.5 1.9 10.3 8.5 12.6 0.9 14.6 0.6 0.732

% Change from 1st day

At 7th day 19.6 42.4 77.6 27.7 -3.6 -

At 14th day 37.3 33.8 75.3 49.4 52.2 -

At 21st day -25.7 -22.5 22.0 -20.7 -100.0 -

At 28th day 46.8 32.4 43.3 40.2 61.4 -

� In group I a suggestive increase is seen by 7th day,a moderate increase by 14th day

and 28th day but there seems to be a decreased % change by 21st day.

� In group II a moderate increase in % by 7th ,14th and 28th day and a significant

decrease by 21st day.

� In group III a moderate increase by 7th day,a highly significant increase % by 14th

day,a suggestive increase by 21st day,a moderate increase by 28th day.

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� In group IV a suggestive increase by 7th day,moderate increase by 21st day,a

significant decrease by 21st day,a moderate increase by 28th day.

� In group V a negligible decrease in TPR% by 7th day, a moderate increase by 14th

day,highly significant decrease by 21st day, moderate increase by 28th day.

Table 51: Comparison of CRT between groups.

Group I Group II Group III Group IV Group V

CRT

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 0.7 0.0 1.3 0.8 1.0 0.5 1.2 0.6 0.9 0.5 0.231

7th day 0.7 0.1 0.9 0.7 0.4 0.2 0.7 0.1 0.6 0.2 0.068+

14th day 1.1 0.6 1.6 0.1 1.4 0.2 1.8 0.7 1.0 0.4 0.038*

21st day 1.3 0.6 1.1 0.7 1.6 0.4 7.1 2.4 1.3 0.8 <0.001**

28th day 3.7 0.0 23.6 0.6 48.9 6.9 33.9 0.1 34.7 0.7 0.462

% Change from 1st day

At 7th day -2.1 -31.3 -62.4 -44.3 -36.5 -

At 14th day 8.5 28.5 38.9 41.2 8.9 -

At 21st day 4.5 -15.5 59.8 74.1 36.4 -

At 28th day 4.1 84.3 71.6 73.6 97.3 -

� In group I a negligible change is seen in CRT change.

� In group II a moderate decrease by 7th day, a suggestive increase by 14th day,a

suggestive decrease by 21st day,a highly significant increase by 28th day.

� In group III a moderate decrease by 7th day,a moderate increase by 14th and 21st

day,a nearly highly significant increase by 28th day.

� In group IV a moderate decrease by 7th day,a moderate increase by 14th day,a

highly significant increase by 21st and 28th days.

� In group V a moderately significant decrease by 7th day,a moderate increase by

21st,highly significant increase by 28th day.

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Table 52:Comparison of BUN between groups.

Group I Group II Group III Group IV Group V

BUN

Mean SD Mean SD Mean SD Mean SD Mean SD

P value

1st day 53.4 13.9 67.8 17.5 61.0 7.5 55.9 14.4 62.5 15.5 0.298

7th day 53.9 12.1 30.9 9.6 55.5 13.4 78.0 35.8 67.3 11.2 <0.001**

14th day 93.2 43.9 101.5 104.2 155.6 18.3 101.5 104.2 63.0 41.2 0.295

21st day 33.3 25.9 201.9 20.0 26.2 20.5 41.2 18.2 32.7 13.5 <0.001**

28th day 127.0 1.4 40.6 21.9 37.5 17.7 113.0 17.0 40.0 16.9 0.006**

% Change from 1st day

At 7th day 0.9 -54.4 -8.9 39.5 77.6 -

At 14th day 74.6 49.7 55.3 81.5 10.9 -

At 21st day -37.7 97.9 -57.0 -26.3 47.6 -

At 28th day 38.0 -40.2 -38.5 100.1 86.0 -

� In group I a negligible change by 7th day,a significant increase by 14th day, a

moderate decrease by 21st day,a moderate increase by 28th day.

� In group II a moderate decrease by 7th day,a moderate increase by 14th day,a

highly significant increase by 21st day,a moderate decrease by 28th day.

� In group III a negligible decrease by 7th day,a moderate increase by 14th day,a

moderate decrease by 21st dayand 28th day.

� In group IV a moderate increase by 7th day,a highly significant increase by 14th

day,a moderate decrease by 21st day, a highly significant increase by 28th day.

� In group V the % increase is highly significant by 7th day,a moderate increase by

21st day,a highly significant increase by 28th day.

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Table 53: Shows the HISTOPATHOLOGY RESULTS :- HEART: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal II Normal Normal Normal Normal III Normal Normal Mild congestion Mild

infiltrations IV Normal Normal Normal Normal V Normal Mild congestion Normal Oedematous

fibres,severe oedema

BRAIN: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal II Normal Normal Normal Normal III Normal Normal Normal Mild congestion IV Normal Normal Normal Normal V Normal Congestion of

cells Mild degenerative changes

Mild congestion,infiltration of few mononuclear cells.

LIVER: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal II Normal Normal Normal Normal III Normal Normal Normal Fatty changes IV Normal Normal Normal Increased kupffer

Cells. V Normal Congestion of

cells Mild degenerative changes

Congestion,haemorrhage Swollen & necrosed Hepatocytes,degeneration

KIDNEY: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal II Normal Normal Normal Normal III Normal Normal Normal Mild congestion

IV Normal Normal Normal Normal

V Normal Normal Severe congestion.

Necrosis,tubular changes.

LUNG: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Mild

congestion Mild congestion

Mild congestion

Mild congestion

II Mild Mild Mild Mild

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congestion congestion congestion congestion III Mild

congestion Mild congestion

Mild congestion

Mild congestion

IV Mild congestion

Mild congestion

Mild congestion

Mild congestion

V Mild congestion

Mild congestion

Severe congestion.

Oedema,mild emphysema

SPLEEN: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal

II Normal Normal Normal Normal

III Normal Normal Normal Normal

IV Normal Normal Normal Normal

V Normal Normal Normal Mild congestion, Haemorrhage.

GASTRIC MUCOSA: GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAY I Normal Normal Normal Normal

II Normal Normal Normal Normal

III Normal Normal Regenerative Changes

More basophilic cells.

IV Normal Normal Normal Normal

V Normal Normal Keratinization Keratinization Slightly proliferating cells.

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DISCUSSION “The great enemy of knowledge is not error, but inertness. All that we want is discussion; and then we are sure to do well, no matter what our blunders may be. One error conflicts with another, each destroys its opponent, and truth is evolved.” - Henry Thomas Buckle Any theories or observations emerged from exhaustive classical and experimental

study can only be accepted if there is a proper reasoning (tarka) of the results &

observations. When we begin with doubts, we shall end with certainties. Therefore, the

theories and facts are put forth for discussion in front of the scientific community. Such

discussions and scholastic deliberations let the scientific fraternity to ameliorate by

knowing both the theoretical and practical profiles about the work.So, here is a sagacious

discussion regarding the in and outs of present study entitled “Pharmaceutico-analytical

and toxicological study of Shuddha Malla on Wistar albino rats (An experimental

Study)”.

It is analyzed under 4 headings.

• Review of literature.

• Pharmaceutical study.

• Analytical study.

• Experimental study.

Review of literature:

Review of literature is discussed under following headings.

∗ Drug Review (Malla, Karavellaka).

∗ Review of Pharmaceutical Procedure.

∗ Analytical Review.

∗ Experimental Review.

Drug Review:

Malla is in use as a rasoushadhi since ages.Eventhough it is highlighted in major

Rasashastra texts, it was first mentioned by Sushrutha as a therapeutic drug. Its wide

applications as medicine in shwasa, kushta, yakshma, sandhivata, phiranga etc., Its

actions as balya, vrushya, rasayana has given it the name. After all these merits Malla has

a synonym of Dhathu Visha/ Shankha Visha with its aushadhi mathra as 1/120th-1/30th

of a ratti wherein the maaraka mathra is also mentioned cautiously to be 1 ratti.Therefore

enough care is taken during Malla matra nirmana as a safety concern.This gives a base to

think about the relationship of dose and toxicity.

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Inorganic arsenic is a human poison. Organic arsenic is less harmful. Arsenic is

an ultra-trace essential element (nonmetal). The deficiency signs are impairment of

growth, reproduction, heart function. The specific function is increased arginine leads to

urea and ornithine which helps in metabolism of methyl compounds.After a very small

dose of arsenic most of the absorbed inorganic arsenic undergoes methylation, mainly in

the liver, to monomethylarsonic acid and dimethylarsinic acid which are excreted, along

with residual inorganic arsenic in the urine. However, if the dose of arsenic is very large,

the elimination half-life is prolonged. Once absorbed, arsenic rapidly combines with the

globin portion of haemoglobin and therefore localises in the blood. In one report from

China the monotherapy of Arsenic trioxide produced a complete clinical response in 9 of

10 with relapsed APL.Consequently, TRISENOX (As2 O3) was approved for the relapsed

or refractory APL by US FDA in September 2000.This gives a clear picture of the

commendable updates from the modern research.

Karavellaka (Momordica Charantia) belongs to Shaaka Varga,Cucurbitaceae

family.It is a tiktha skandha dravya, widely grown for edible fruit, which is among the

most bitter of all vegetables.Classically meant to be tridoshashaamaka.Its main actions

are said to be to kill bacteria,viruses,cancer cells,leukemia cells,prevents tumors,fights

free radicals,cleanses blood,detoxification etc.,which would not only help the purification

procedure but also enhance the drug qualities to which it comes into contact.

Glycosides,Alkaloids, charantin, charine, hydroxytryptamines, lanosterol, rosmarinic

acid, rubixanthin, spinasterol, steroidal glycosides,stigmasta-diols etc.Galactouronic acid

which is found as a rare and special entity can complex with the toxic molecules for the

organization, may help in the obviating and eliminating the toxic properties of Malla

which might be making the drug molecule assimilative and enhance for its prerogative

action.

Review of Pharmaceutical Procedure:-

Pharmaceutical procedure adopted in the present study was Shodhana samskara

by “Swedana”

• “Samskara” which defines itself as ‘Gunantharaadhaanam’ i.e

enhancement of the qualities of a drug and ‘Doshaapanayanam’ i.e to

remove the unwanted impurities.

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• “Shodhana” is one of the initial norms to be instigated in any of Rasa

preparations without which it remains unaccomplished. The word meaning

may merely be the Purification but unto its depth it incarcerates the toxic

substances and enhances the efficacy and safety of the drug molecule.

The mode of each pharmaceutical procedures are unique in its own way.

• “Swedana” is one of such ascendant procedures adopted which is used for

shodhana of many Rasadravyas. In this process the drug is boiled in the

liquids, which are ksharas, amlas or both, and medicinal juices, with the

help of Dolayantra. Fluxation process would have occurred in this kind of

Shodhana.

Now a days some of the Rasadravyas are artificially prepared. So shodhana is

indicated to eliminate all such toxic qualities, induce and enhance certain special qualities

which are essential for the easy assimilation of the material in the living body.

• Dola Yantra and Khalva Yantra are used instruments which are the

basic engineered techniques for purification method.

Analytical Review:

The Physico – Chemical tests like Ash value, Acid insoluble ash, Water insoluble

ash, Loss on ignition, Loss on drying and pH value are some of the physicochemical

tests. AAS for the estimation of Arsenic. AAS is a rapid method if only a few elements

are being tested. X-ray diffraction is a novel and a powerful technique in the elucidation

of 3-dimensional structure, composition of any matter. Structural features can be

determined at molecular and atomic level. It is an important technique for establishing the

batch-to-batch reproducibility of a crystalline form is X-ray Powder diffraction.Particle

Size analysis through laser diffraction technique is one of most upgraded technique which

has a wide dynamic measuring range, Flexibility, Generation of volume-based particle

size distributions, Rapid data acquisition, Ease of Verification.

Toxicological review:

'Toxicology' traditionally known as the 'science of poisons' began with early

cave dwellers who recognized poisonous plants and animals and used their extracts for

hunting or warfare. Simultaneously, with time, to determine the effectiveness of a

particular compound the concept of toxicology was developed.

Toxicology basically is defined as the study of the effects of chemical agents

on biological material with special emphasis on the harmful effects. After gaining

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relevant information on the harmful effects of a compound the levels for its safe usage or

the degree of its safeness is established, this is also known as its (compound) Biosafety

level. A central concept of toxicology is that effects are dose-dependent; even water can

lead to water intoxication when taken in large enough doses, whereas for even a very

toxic substance such as snake venom there is a dose below which there is no detectable

toxic effect.

Experimental Review:

The materials and methods of the study or the study design in which the

rationale of the Species selection,animal house facility,age of rats,steps to be followed

before while and after experiments,group sizes,route of administration,dosage

schedule,acute and subacute toxicity methods,measurements and observations to be

made,laboratory parameters, terminal studies and statistical methods are reviewed wide

infra to be updated in the procedural concepts.

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DISCUSSION ON PHARMACEUTICAL METHODS:- “The nature of the chemical bond is the problem at the heart of all chemistry”

Discussion on Malla Shodhana:

About 50 gm made into small pieces of Malla was tied in a clean, white, square

shaped kora cloth measuring 10 inches in length and 10 inches in breadth.A strong

thread was used to tie the pottali and this pottali was suspended in the pot containing

Karavellaka swarasa (pH 6) using a wooden rod. Mild heat (Mrudvagni) was given for

6hrs (2 yamas). Karavellaka Swarasa was added as and when required to maintain the

minimum liquid level. After 6hrs (2 yamas), heating was stopped and the contents were

allowed to self-cooling. Then the pottali was removed from the liquid and washed

thoroughly with hot water.After shodhana Malla appears dull white,opaque solid.

The above method of shodhana can be rationalized in following ways:

� Size of the compound particles:

The Malla is broken into pieces before placing it for shodhana. As the perimeter

of the bigger sizes is in larger units. The acid particles can only collide with the edge

of the chip. However, if we break up the large chip into smaller chips. Notice how the

acid in the second diagram can reach what used to be the centre of the large chip.

Reducing the size of particles increases the rate of a reaction because it increases the

surface area available for collisions to take place. This increases the number of

collisions. It has no effect on the energy of the particles.

Fig 62 : Shows the compact and fragmented particles.

� Why Pottali? Malla is tied in a Pottali as Arsenic trioxide is highly volatile and

because of its O2 content which is paramagnetic, therefore to prevent this reaction

with the external environment,it is tied in layers of cloth having minute pores for

the swarasa to enter.

130

� Spatial order: Two boxes of materials separated by a membrane so that the

material can diffuse between the 2 boxes. The pottali made of cloth which acts as

a membrane is allowed to swing inside the hot juice bath so that the collision

between the compound and the solution takes place from all directions.

� Ion exchange : Ions between a solution and a solid highly insoluble body in

contact with it. The solid (ion exchanger) must of course contain ions of its own,

for the exchange to proceed sufficiently rapidly, the solid must have an open

permeable molecular structure so that ions and solvent molecules can move freely

in & out.Malla the solid molecule allows such a process.Karavellaka swarasa acts

as a chelating ion exchanger in which the chelating groups are incorporated and

are attracted to the Malla matrix as interionic effects are not negligible even for

weak acids. Therefore a complex ion is formed by the union of a simple ion with

either other ions of opposite charge/neutral molecules.Since Phosphorous is a

constituent of Karavellaka swarasa it inhibits the trimethylarsine formation,a toxic

metabolite.Infact ‘P’ also helps in arsenic uptake to the target site.

� Why 6 hours? Law of Mass action:In Chemical kinetics the Speed of the

chemical reaction is proportional to quantity of reacting substances in an optimum

time.

Three major modes of complex series of Arsenic biotransformation in the

environment are redox transformation between arsenite and arsenate,the reduction

and methylation of Arsenic, ligand exchange,biosynthesis of organoarsenic

species which is obtained in an optimum time and temperature.

� Karavellaka pH 6 : The swarasa is a weak acid and the degree of dissociation

of most arsenic species is strongly influenced by the pH.Arsenic is present in the

undissociated state in acidic solutions and in solutions not exceeding pH 8.Also a

weak Van-der Waals interaction between the unwanted metal impurities is

facilitated to eliminate them from the drug Malla to make it pure.

The effect of temperature:-

� When we increase the temperature at which a reaction is taking place, the

particles move more quickly. This has two effects:

1) More collisions take place.

2) When a collision occurs, there is more chance that the collision will lead to a

reaction, because the amount of energy is more likely to be greater than the

131

minimum amount of energy needed (the activation energy).{Maxwell-Boltzmann

distribution of molecular energies}.

Fig: 63 Shows the effect of temperature and reaction process.

� Activation energy is the minimum energy required before a reaction can occur is

the energy for collision reaction. It is involved in breaking some of the original

bonds. This is the cause of agni sannikarsha for the shodhana process.

� The 'rule of thumb' that the rate of chemical reactions double for every 10 °C

temperature rise is the reason why agni is increased slowly from mrudu to madya

and then teekshna.

� As the time and temperature is increased the consistency of boiling swarasa

changes to a thin liquid and later a foamy substance appears on the rim of the

pot.According to 2nd law of thermodynamics the entropy of the physical or

chemical system and its surroundings must increase with time i.e orderly energy

of a system like uniform motion must degrade eventually to the random motion

particles in heat bath.This physio-biological treatment makes Malla biologically

compatible.

The effect of concentration:

Fig: 66 Shows the effect of concentration on collision.

� Increasing the concentration of a solution leads to more collisions (greater

frequency of collisions) so the rate of the reaction goes up. Karavellaka swarasa is

added repeatedly to allow high concentrations resulting in high rate of reactions.

132

� Autocatalytic reactions where chemical reactions in which at least one of the

products is also a reactant i.e the product Malla is a reactant and a catalyst. The

proteins , steroids and enzymes in karavellaka swarasa also act as catalysts.

� It can be considered as an elimination reaction in which a type of organic

reaction in which substituents are removed from a molecule in either a one or two

step mechanism.

� Biosorption: At a maintained pH , a constant raising temperature and the speed of

particles the organic solution absorbs the metal content from the compound drug

known by the difference in initial concentration and the metal ion remaining in

solution.This gives an idea of the absorption of unwanted impurities of Fe,Sb etc

metal ions being absorbed from the compound Malla making it ultrapure.High

temperatures wouldn’t allow such reactions to take place.

� Organic encapsulation : The drug molecule Malla is made safe and bioavailable

when there is an attempt to prepare and characterize a carrier system to minimize

the toxicity of the drug, retention of the drug and enhances a sustained release like

liposomes.

� Drug designed to target: Malla is made more assimilable through organic

reactions to the system and signalling it to the target.

� Safe ADME :- As inorganic As compounds have rapid oral absorption the

organic innervation ,conversion into methylated form paves a way for safer forms.

133

TO DISCUSS ON ANALYTICAL STUDY

Analysis of the drug is necessary to know the physico – chemical, macro and

micro properties and to confirm the purity of the drug. According to ancient parameters

the varna of Malla was changed from Pure white to dull white,its crystal glassy

appearance was unseen.It shows the reduction in crystallanity..

Physicochemical analysis:To ensure, the essential components, which are present within

a predetermined range of composition, physicochemical analysis is necessary.

Discussion on Ash value: It is a physical method useful in drug standardization. Ashing

involves an oxidation of the components of the product. It gives a percentage of inorganic

constituents of the sample. It also helps in judging, identification of sample or purity of

the drug. The two samples of Malla i.e. Ashuddha Malla and Shuddha Malla were

evaluated for ash value and it was found to possess 8.92% and 8.86% respectively which

indicates that Shuddha Malla contains less Ash value and more amount of bio human

available particles. Hence it can be said that, the Shodhita Malla is within the standard

limits.

Discussion on Acid insoluble ash: The human metabolic process and pharmaco kinetics

depends upon purity and human acceptable forms of drug. When the ash of the two

samples Ashuddha and Shuddha Malla were mixed with Hydrochloric acid, 0.77% and

0.48% amount of acid insoluble ash were detected which are not soluble and digestible in

human GI tract. As Shodhita Malla contains least of silica etc and insoluble ash material,

which can also be ascertained that in the present study the Shodhita Malla dwells in the

drug standard tests and hence safe. Hence it can be said that, the Shodhita Malla are

within the predetermined range.

Discussion on water soluble ash: Negligible differences are seen in the determination

of water soluble ash and as it is insignificant in case of inorganic substances, Malla has

shown negligible values before and after shodhana.

Discussion on loss on drying at 1100C: The moisture content of any pharmaceutical

agent spoils not only the drug activity but also everything. Loss on drying at 1100C is a

physical test to detect the percentage of moisture content and hence the shelf life of the

sample. Lesser the loss on drying at 1100C, the better will be the drug. In the present

study, the two samples of Malla i.e. Ashuddha and Shuddha Malla were found to posses

134

0.09% and 0.12% loss on drying at 1100C. Hence it can be stated that it possess least

moisture content and hence very rare chance of bacterial and fungal growth and also the

drug is having least or nil hydroscopic activity and the drug deterioration chance or

contaminations chances etc. are very less. Concurrently it can be stated that the shelf life

of the Shodhita Malla in the present study is more and are within the predetermined

range.

Discussion on Loss on ignition:Loss on ignition of Ashuddha Malla was 82.84% and

Shuddha Malla was 81.39%.Its a known fact that Arsenic is highly volatile and

sublimates easily into air. But still this test shows that even at high temperatures Shuddha

Malla is having less amount of loss and Ashuddha Malla shows more amount of loss. It

shows that after shodhana the drug is more stabilized.

Discussion on pH: The pH value was estimated for the two samples of Malla i.e.

Ashuddha and Shuddha Malla. The pH of the two samples was found to be 6.98 and 7.08

respectively. The Ashodhita Malla is within acidic range or it is a weak acid. But the

Karavellaka Shodhita Malla revealed alkalinity or can be considered a weak base. The

weak acid and a weak base form a neutral pH which is the need for suitable biologic

assimilation.

Discussion on Arsenic estimation: The two samples of Malla i.e. Ashuddha and

Shuddha Malla when subjected to AAS for the estimation shows that it contains

qualitatively and quantitatively 69.7% and 74.22% of Arsenic (As%) respectively. The

genuine drug taken from the market is confirmed for its quality i.e Ashuddha Malla.The

percentage of Purity is increased in case of Shuddha Malla which is the sign of potent

drug without any other trace metals & impurities that might have adsorbed and detoxified

by Karavellaka swarasa.

Discussion on X- RD:-.

According to X- RD patterns composition of Ashuddha Malla i.e. 1st sample is

As2O3 or Arsenolite which is confirmed only after comparing the d-identified peak values

which are like fingerprints of the particular sample with d-standard peak values. The

standard sample which was compared to Ashuddha Malla sample consisted Fe,Mg,Sb,Si

0.001 to 0.01% each.Ca and Pb traces and was Isostructural with senarmontite.This gives

a clear view of the sample containing impurities which would match to the standards.

Composition of 2nd sample i.e. Karavellaka Shodhita Shuddha Malla is As2O3,

Arsenolite which is confirmed after comparing d-identified peak values with d-standard

135

peak values.The standard which was compared to Shuddha Malla sample consisted of no

such impurities and was a pure Arsenic trioxide.Analyzing the above two factors we can

assume the biological purification method which is undertaken due to Shodhana.

Though there is slight shift in the peaks between both the samples it is significant

as it shows such a difference.The d-spacing values might be in a second decimals

difference but it also suggests the change after shodhana with the innervation of media

and process.

The crystal lattice structure of both the samples were Face Centered Cubic but

only the difference was in the unit cell volume between the two.This Change also is a

sign of stoichiometric differences after shodhana.

The count of the Ashuddha malla is about 6858 and that of Shuddha Malla is

about 4771 which signifies the reduced crystallanity of the latter after Shodhana

procedure. The pharmacological need is the ease of drug ADME, lesser the crystallanity

better is the absorption.

Discussion on Particle Size: The Particle Size was done using Laser Diffraction

technique which is the latest version of analysis.The particle size of Shuddha Malla after

Shodhana has increased which indicates its organic encapsulation to target the

microsphere drug molecule for safe slow and sustained absorption.As particle Size and

particle size distribution can influence the bioavailability,principally by changes in

surface area of the drug which is exposed to the acid degradation it is one of essential

factors.

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Discussion on ACUTE STUDY OBSERVATIONS:-

� The behavioural profile shows the awareness of the rats initially and later its

passivity shows depression. Restlessness shows the mood of the animal and the

touch response shows its motor activity.

� The neurological profile shows central excitation, motor in co-ordination, muscle

tone and reflexes.

� The autonomic profile shows the muscarinic activity which is a sign of sympathetic

stimulation.

� The general sign shows CNS excitation/sympathomimetic action.The reduced food

intake is may be due to chronic pain.The retreat to the corner of the cage are

examples of distress in anticipation of an experimental procedure.

Summarizing the Profile data when evaluated shows the basal score of all

other groups to be normal except group IV and VII which shows a abnormal sign

which is a sign of absolute toxicity in higher escalated doses.

Discussion on Body weight:-

� Considerable decrease (less than 5%) in body weight of the rats were noted by 3rd

day of dose administration in all the test groups except control may be because of

the drug Arsenic trioxide (Malla) which acts in detoning the muscular protein mass.

� Group IV and VII have a significant decrease in body weight which shows that the

condition is deteriorating due to escalated doses is a warning of the preceding death.

� Though group VI shows a suggestive decrease in weight, group III seems to be

stable i.e though the dose of Ashuddha Malla & Shuddha Malla are similar this

difference is considerable.

� Significant weight gain in all groups by 12th-14th day is seen except in group VII

that cannot be noted because of the death. This may be because Arsenic Trioxide

has the threshold effect after certain doses.

Discussion on Impending Death :-The rats which were dead in group IV

and VII had an impending death as they were in moribund state or death is expected

prior to the next planned time of observation. Signs indicative of this state in rodents

included convulsions, recumbency, and tremor.

� Even the 10,20,30 and 35 times the therapeutic dose of Shuddha Malla shows no

toxic sign.

� The 25 times the therapeutic dose of Ashuddha Malla is toxic.

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� The median lethal dose of Shuddha Malla is 16.65mg where as that of Ashuddha

Malla is 12.96mg which shows the variable dose significance due to Shodhana.

� The humane end point was seen at the dose of 14.4 mg of Shuddha Malla whereas

at the dose of 10.8 mg in case of Ashuddha Malla.This shows the vitality of the

Shodhitha Malla as a Safer drug.

� The first point along the graph where a response above zero is reached is usually

referred to as threshold-dose,therefore the threshold dose of Shuddha Malla is

around 12.6mg where as that of Ashuddha Malla is about 9mg.It is also a mark of

evidence for the Shoditha Malla to be Safer.

� The stronger a particular substance is,the steeper the curve will be.Between the two

Samples Shuddha & Ashuddha Malla the latter is more steeper.

Discussion on Dose-response relationship:

� Greater the therapeutic ratio, Safer is the drug.The therapeutic index of the drug

Shuddha Malla is greater when compared to that of Ashuddha Malla helps us to

make the risk-benefit comparisons between the potential drugs.

� According to some research the no toxic effect dosage obtained in a rodent toxicity

may be divided by 10 which is equal to the maximum dosage for clinical use.

Therefore the Shuddha Malla safe dose may be 12.6÷10= 1.26mg which falls into

the classical dose range.

� The therapeutic ratio must be atleast more than one for it to be safe. Though both

Shuddha & Ashuddha Malla indices are greater than one and proves to be in Safer

limits at the prescribed therapeutic drug doses Shuddha Malla is significantly better

& safer than Ashuddha Malla and to the degree to which the clinical dose schedule

may be varied.

Discussion on Blood Parameters:-

Haemoglobin (Hb):

� The marginal decrease in all the groups except in control & that with escalated dose

of Shuddha Malla might be because of the degradation of fusion protein due to the

interaction of ATO.

� The significant decrease is a sign of anaemia which is observed with a single dose of

Shuddha & Ashuddha Malla is because haemodilution action of ATO.

� As a compensating process when Shuddha Malla is administered in repeated doses it

has shown a marginal increase which is a sign of normal regeneration.

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� As ATO always has an affinity towards the blood proteins, the concentration of Hb%

is increased in a faster rate in escalated doses group when compared to others.

� Comparing daily therapeutic doses of Ashuddha & Shuddha Malla the Hb% decrease

is to lesser extent in the latter.

� The percentage of increase shows the oxygen carrying capacity being stimulated

which is seen in Shuddha & Ashuddha Malla daily therapeutic doses where the

increase is much better in the former.

� Decrease in Hb in the Ashuddha Malla escalated dose reveals the form of aplastic

anaemia due bone-marrow depression.

Total Leucocyte count :

� Leucocytosis is observed in Ashuddha Malla escalated group in 10.8mg dose which

may be the result of disruption of margination of leucocytes along the vascular

endothelium.

� Leucocytosis is also observed in Shuddha Malla escalated dose of about 23.4mg.

Differential Counts :

� Moderate increase in polymorphs control may be due to stress related to experimental

procedures and environmental conditions.

� An abrupt increase in polymorphs is seen in Ashuddha Malla daily therapeutic dose is

a sign of toxicity.

� Though a negligible decrease is seen in lymphocytes it is a sign of

immunosuppression in all the groups due to experimental stress related.

� A marginal increase by 14th day at 23.4mg (65 times the therapeutic dose) Shuddha

Malla group is a sign of lymphocytosis.

� An eosinophilic condition exists in the Shuddha malla & Ashuddha malla escalated

doses wherein which the the condition is better in the former drug.

� Even in single doses of Ashuddha & Shuddha Malla eosinophilia is seen but the latter

being better than former.

� The daily therapeutic doses of Ashuddha & Shuddha Malla shows a significant

difference where it is only a negligible increase in eosinophils in Shuddha Malla daily

doses but an absolute eosinophilic condition in daily doses of Ashuddha Malla may

be because the recovery process happens in the former but not in latter.

� Ashuddha Malla at 25 times & Shuddha Malla at 45 times the therapeutic dose

group shows Monocytopenic condition in escalated doses may be due to bone marrow

failure and aplastic anaemia.

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Packed Cell Volume:

� The increased haematocrit condition in control may be due to haemoconcentration

and decrease in haematocrit in all other groups may be due to hemodilution and

expansion of plasma volume.

� In Shuddha Malla daily therapeutic doses negligible increase is observed by 14th day

after decrease by 7th day reveals a recovery or compensation of RBC counts.

� The decreased RBC count in Shuddha Malla & Ashuddha Malla escalated doses, a

single dose and repeated doses of Ashuddha Malla may be due to haemodilution and

anaemic condition.

Discussion on Serum Parameters :-

� Increase in Serum urea in both the escalated groups of Ashuddha 35 times & Shuddha

Malla 60 times is a sign of acute renal failure,azotaemic condition.

� Ashuddha Malla at 20 times and Shuddha malla at about 55 times the therapeutic

doses shows the moderate increase in creatinine levels which is a sign of impaired of

renal function.

� Ashuddha Malla in escalated doses of 25 times the therapeutic dose has shown

increase in SGOT levels which signifies increase in tissue injury to liver as well as to

other tissues like in myocardial infarction.

� A marginal increase in 55 times the therapeutic dose of Shuddha Malla, a single dose

and the daily repeated dose of Ashuddha Malla reveals minor enzymatic changes.

� Ashuddha Malla at 30 times therapeutic dose with highly significant increase in

SGPT which is specific for liver tissue due to liver cell injury. The same condition

exists in Shuddha malla escalated doses but in the dose of 65 times the therapeutic

dose which shows mild enzymatic changes.

Discussion on Histopathology :-

Normal architecture of all organs are seen in Control group, single dose of

Shuddha Malla and Ashuddha Malla except the lung architecture which has a mild

congestion due to ether anesthesia before decapitation or sacrificing. There is a mild

lesion in gastric mucosa in the group of Shuddha Malla daily therapeutic dose which may

be due to irritant action of the drug.

The escalated doses i.e the 65 times therapeutic doses of Shuddha malla have

shown the toxic markers where there is destruction of cardiac fibres , mild congestion and

degenerating neurons in brain,mild fatty infiltrations of liver with severe

haemorrhage,mild congestion and degeneration in kidney tissues,lungs with

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oedema,haemorrhage,infiltraton of inflammatory cells,mild congestion in spleen,gastric

mucosa with mild keratinization and descumated krypts.

With repeated daily doses of Ashuddha Malla there is congestion of cells in

heart,mild lesion and haemorrhage in brain,periportal infiltration of liver,atrophic

glomeruli in kidneys,severe haemorrhage around the vessel in lung tissues,destruction of

villi in gastric mucosa which are the absolute markers of toxicity.The escalated doses i.e

the 30 times therapeutic dose of Ashuddha malla shows a severe

haemorrhage,destruction in cardiac fibres,specially with periartirits of lungs,proliferation

of lymphoblasts in spleen show the signs of a highly toxic drug than Shuddha Malla in

escalation.

Discussion on SUB- ACUTE STUDY OBSERVATIONS: The behavioural profile shows the awareness of the rats,hyper activity is due to

the CNS stimulation which is observed in repeated doses of Ashuddha and Shuddha

Malla.The colour change in faecal matter may be due to excessive output of inorganic

ATO after the required assimilation.

Discussion on Body weight:-

� As a state of drug interaction on muscle tissues where ATO has its affinity

towards protein groups the decreased weight is seen in all groups except control

by 3rd day.

� There is a failure to gain body weight in repeated doses of Ashuddha Malla

when compared to Shuddha Malla repeated doses which is a sign of an adverse

effect.

� Even with a single dose of the test drug there is a variance between the

Ashuddha and Shuddha Malla where the latter is faster in recovering or gaining

weight.

Discussion on Blood Parameters:-

� Since the drug ATO has a direct effect on capillary permeability there is a

suggestive significant decrease of Hb in groups of single doses of Shuddha &

Ashuddha Malla.

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� Though the haem-concentration is compensated in faster rate in repeated dose of

Shuddha Malla it would take still later days in case of Ashuddha Malla.

� Leucopenia is observed in repeated doses of Ashuddha malla within 14-21 days of

drug schedule which is not seen in Shuddha Malla doses which is a significant

change .This may be because of the form of the drug encapsulation made organic

and safer for absorption.

� No comparable changes are observed in DC- polymorphic changes.

� Repeated doses of Shuddha Malla shows a constant slower rate of decrease in

lymphocytes whereas the Ashuddha Malla has an abrupt decrease which is the

sign of immunosuppression in a faster rate.

� A descending type of eosinopenia is seen in Ashuddha Malla single dose where

this sign is seen only by 28 days in case of Shuddha Malla.

� An absolute eosinopenia is seen in Ashuddha Malla daily therapeutic dose by 14th

day which is a sign of decrease in ACTH or acute infections which is unseen to

this extent in case of Shuddha Malla.

� The monocytosis stage is observed which suggests of any vascular disease in

Ashuddha Malla repeated doses of Ashuddha Malla which shows a linear increase

as days pass on but in Shuddha Malla though there is such a stage it compensates

within next few weeks.

� The highly significant decrease in RBC in Ashuddha Malla repeated therapeutic

doses is a sign of lesser number of circulating red cells which gives a gross

picture of the toxic effects on haemopoiesis or matured circulating cells.

� The haematocrit value has a negligible change and therefore not of much

consideration.

Discussion on Serum Parameters:-

� A highly significant increase of SGOT in a faster rate due to Ashuddha Malla

signs an abnormal liver tissue and necrosed cells, also the cardiac tissue

injury.

� There may be mild enzymatic changes in case of single doses of both the drug

samples as ATO reacts rapidly with enzymes.

� Both the single dose and repeated doses of Ashuddha Malla shows a increase

in SGPT by 21-28 days is a suggestive sign of liver toxicity.

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� The SGPT increase in Shuddha Malla single and repeated doses is less

compared to that of Ashuddha Malla.This shows the difference of the drug

pharmacology.

� The decrease in albumin in single dose of Shuddha Malla and Ashuddha

Malla shows may be due to haemconcentration.

� The increase levels in albumin in repeated doses of Shuddha Malla is a sign

of maintenance of osmotic pressure needed for proper distribution of body

fluids between intravascular compartments and body tissues.

� Though there are similar levels seen in case of Ashuddha Malla initially there

are fluctuations in later days which otherwise is a sign of disturbance in

circulation of body fluids.

� The increase in repeated doses of both Shuddha Malla by 28th day &

Ashuddha Malla by 14th day is a sign of glomerular filtration rate though the

latter seems to be more nephrotoxic or renal insufficiency.

� Repeated doses of Ashuddha Malla showing a sign of increase in BUN shows

the is a marker of renal function and the state of anti-diuretic hormone.

Discussion of Histopathology results:-

� Repeated dose of Ashuddha Malla showing the oedematous fibres ,severe oedema

in heart is a sign of intense toxicity leading to myocardial damage, when

compared to that of Shuddha Malla which shows only mild changes in tissue.

� Congestion of brain tissues and infiltration of few mononuclear cells is a sign of

toxicity of repeated doses of Ashuddha Malla in repeated doses which had already

started by 14th day.

� Highly toxic is the state of liver tissues especially to the hepatic parenchyma in

repeated doses of Ashuddha Malla where this process has started by 14th day.

� Even with single dose of Ashuddha Malla a negative sign is seen on 28th day with

increased kupffer cells.

� Mild fatty changes of Liver in repeated doses of Shuddha Malla may be due to

specific pharmacology in rats for As.

� The tubular changes and necrosis in kidney tissues is a sign of absolute toxicity

due to Ashuddha Malla repeated doses where severe congestion is already seen by

21st day where these signs are absent in case of Shuddha Malla is a good sign of

positive approach of the processed drug.

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� Mild congestion of lungs is very common in all groups as seen which is because

of the procedural effect i.e the ether anaesthesia which is a sign of stress on lungs

and the respiratory system.

� By 28th day mild congestion and haemorrhage of spleen in Ashuddha Malla is the

sign of toxicity to haemopoetic system .

� Repeated doses of Ashuddha Malla shows a toxic profile of gastric mucosa due

to keratinization by 21st day but the positive fact by the same period in case of

Shuddha Malla is the regenerative changes or more of basophilic cells seen in

tissues of gastric mucosa.A sort of replenishing and recovery process is seen at

this stage.

In a nut shell as the metabolites of ATO have uncertain biological

effects it is still an unraveled fact about its hit target mechanism.The methylation

of ATO rapidly to dimethylsarsenic acid and hepatic methylases helps the

mechanism of metabolic detoxification.Research studies show that an i.p dose of

2mg and oral dose of 5 mg results indicate no toxicity. The methyl transferases

involved in the biomethylation of As have interesting properties since they have a

“hidden” redox component..The administration of the drug Shuddha Malla with

specific anupanas which may act as adjuvants or synergists may take the drug

molecule safely. As rasoushadies classically has been adopted for a

mandala/ardha mandala as per some experts it is a brilliant way of tackling with

the potential toxic drugs. Shuddha malla maintained as a consolidation therapy

would definitely prove its efficacy safely. Though 125mg is mentioned as a lethal

dose, when MTD of rat’s dose extrapolated to humans it is about 140mg for the

sample taken which shows that our seers were careful enough in indicating even

the lethal dose.

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CONCLUSION

� Malla is a drug with wide therapeutic indications with the least dose mentioned in

classicals and also mentioned cautiously 1 ratti to be the maaraka matra.

� As a part of purification and Safe drug potentiating mechanism the

pharmaceutical procedure is the Swedana using Karavellaka Swarasa.

� Quantitative analysis of the drugs Ashuddha & Shuddha Malla reveals more

percentage of purity of Arsenic trioxide in the latter.

� From X-RD study, composition of both the samples Malla was Arsenolite (As2O3)

but identified by two different JCPDS standards.

� The Particle Size of the Shuddha Malla is more than Ashuddha Malla due to

organic innervations after shodhana.

� The median lethal dose of Shuddha Malla is 16.65mg where as that of Ashuddha

Malla is 12.96mg which shows the variable dose significance due to Shodhana.

� The 5 times the dose(1.8mg) of therapeutic dose (0.36mg) proves Shuddha Malla

to be non-toxic in rats.

� The 10 times the dose (3.6mg) of therapeutic dose proves Shuddha Malla to be

non-toxic in rats.

� The 20 times the dose (7.2mg) of therapeutic dose of Shuddha Malla shows no

toxicity.

� The 30 times the dose (10.8mg) of therapeutic dose of Shuddha Malla shows no

toxicity.

� The 35 times the dose (12.6mg) of therapeutic dose of Shuddha Malla shows no

toxicity.

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� The 40 times the dose of therapeutic dose i.e. about 14.4 mg of Shuddha Malla is

a toxic dose in rats.

� In the repeated therapeutic doses for 14 days Shuddha Malla is non-toxic whereas

Ashuddha Malla proves to be toxic.

� In the 28 days repeated therapeutic dose Ashuddha Malla is highly toxic, though

Shuddha Malla shows some of considerable differences in liver architecture, the

regenerative changes proves it to be non-toxic.

� The therapeutic index of Shuddha Malla is 3.08 which prove Shuddha Malla as

non-toxic.

� For the sample of Shuddha Malla taken the maximum tolerated dose is 12.6mg for

rats.

� Shuddha Malla is a very safe non-toxic drug in the classically indicated dosage

limits as per acute and Sub-acute repeated dose studies.

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SUMMARY

The present study entitled “Pharmaceutico-Analytical and toxicological study of

Shuddha Malla on Wistar albino rats (An experimental study)” - summarized briefly

in Literary, Pharmaceutical, Analytical and Experimental parts.

Objectives: 1) Shodhana of Malla by swedana method.

2) Physico – chemical Analysis of 2 samples of Malla i.e. Ashodhita

Malla and Karavellaka Shodhita Malla.

3) To find the median lethal dose of Shuddha Malla.

4)To screen the probable toxicity of Shuddha Malla in repeated doses.

5) To screen the probable toxicity of Shuddha Malla in a single dose.

Introduction:

This part reveals the need for study, aims & Objectives and hypothesis behind

selecting Malla for the toxicity study.

Literary study:

It comprises the literatures on Malla described classically in Rasa texts and also

modern information of Malla (Arsenic Trioxide). It also includes review on the

associated Dravyas used for shodhana such as Karavellaka..

Pharmaceutical review reveals the concept of Shodhana, Swedana along with the

Yantras like Dolayantra and Khalva Yantra, used in present study.

Analytical review includes Physico – Chemical, Analytical methods like Total

Ash Value, Acid Insoluble Ash,Water insoluble ash, pH, Loss on Drying at 1100C ,Loss

on ignition ,XRD,AAS and Particle Size.

Review of literature also includes the concept of toxicology and the current up

dated literature regarding its regulatory guidelines.

Experimental review reveals the study protocol, Albino rats and its behavior,

housing, feeding, environment and also drugs and equipments used in Experimental

study.

Methodology:

Pharmaceutical study:

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It reveals Shodhana of Malla using Karavellaka Swarasa by swedana procedure for 6

hrs.

Analytical study:

Analysis of 2 samples of Malla i.e. Ashuddha Malla and Karavellaka Shodhita

Shuddha Malla . Ashuddha Malla was Pure white and glassy.The Shuddha Malla is dull

white and opaque. Minimum weight loss occurred after Shodhana.

Modern physical tests were conducted on 2 samples of Malla i.e. Ashodhita Malla

and Karavellaka Shodhita Shuddha Malla such as Ash value, Acid Insoluble Ash,Water

insoluble ash, Loss on Drying at 110 ° C,Loss on ignition and pH. The results were as

follows.

Ash values of 2 samples of Malla: Ashuddha Malla – 8.92%, Shuddha Malla –

8.86%.

Acid Insoluble Ash values of 2 samples of Malla: Ashuddha Malla – 0.77%,

Shuddha Malla -0.48%.

Water insoluble Ash values of 2 samples of Malla : Ashuddha Malla – 0.04%,

Shuddha Malla -0.05%.

Loss on Drying at 110°C values of 2 samples of Malla: Ashuddha Malla -

0.09%, Shuddha Malla - 0.12%..

pH values of 2 samples of Malla: Ashuddha Malla - 6.98, Shuddha Malla - 7.08.

Quantitative analysis for As% of 2 samples of Malla i.e. Ashodhita Malla and

Shuddha Malla were 69.72% and 74.22% respectively.

X-Ray diffraction studies were conducted on 2 samples of Malla i.e. Ashodhita

Malla and Shuddha Malla and composition of both the samples was Arsenolite (As2O3),

which is confirmed only after comparing d-identified values with d-standard & peak

values with the face centered cubic crystal lattice.

The Particle Size analysis by Laser Diffraction technique shows the size of

Ashuddha Malla as 23.67µm and Shuddha Malla as 43.32µm.

Experimental study:

Acute Study:-

The Study period was for 14 days. Seven groups of 8 animals in each group.

The groups were Control, single dose of Shuddha Malla, Single dose of Ashuddha Malla,

repeated therapeutic dose of Shuddha Malla, repeated therapeutic dose of Ashuddha

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Malla, escalated doses with multiples of 5 for each day for 14 days of Shuddha &

Ashuddha Malla to find the median lethal dose.

Evaluating the statistics of body weight, haematology & blood Chemistry,

viewing histopathology parameters it shows that the escalated doses have proved absolute

toxicity. The median lethal doses of Shuddha Malla and Ashuddha Malla are 16.65mg

and 12.96mg of respectively. The 10, 20, 30 and 35 times the dose of therapeutic dose of

Shuddha Malla proves to be non-toxic. In the repeated daily therapeutic doses for 14 days

Ashuddha Malla has toxic signs which are not found in Shuddha Malla.

Sub-acute Study:-

The study period was for 28 days. There were 5 groups with 8 animals each.

The Control, Single dose of Shuddha Malla, Single dose of Ashuddha Malla, repeated

therapeutic dose of Shuddha Malla and Ashuddha Malla for 28 days.

The body weight, haematology, blood chemistry and histopathology parameters

show that in the dose schedule of 28 days, considerable toxic sign is found only in liver

architecture in repeated daily therapeutic dose of Shuddha Malla.In repeated therapeutic

daily dose of Ashuddha Malla is proven to be highly toxic accounting all the parameters.

The noticing factor is that the Shuddha Malla in repeated daily therapeutic doses shows

regenerative changes which are an auto-defense mechanism of the drug and body

interaction. In a nut shell Shuddha Malla is a non-toxic safe drug as per drug dose

mentioned (1mg-4mg) in classicals.

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SCOPE FOR FURTHER STUDY

� Similar studies can be done with other Shodhana procedures of Malla.

� Sub-chronic for 90 days & chronic toxicity tests for 6months should be carried

out to disprove toxicity claims.

� Comparative study can be done by taking all similar and dissimilar kinds of

Shodhana procedures of Malla (swedana, Pachana, Putapaka, etc).

� Comparative Analytical, Experimental and Clinical study can be tried for

Shuddha Malla and Malla Bhasma.

� To analyze the structure of Shuddha Malla, NMR spectroscopy, Mass

spectroscopy etc can be studied.

� Comparitive toxicology study of Shuddha Malla & Malla Bhasma prepared by

various methods can be taken.

� Secondary Markers, such as levels of insulin or activated T-cell populations can

be used to study the toxicity of Shuddha Malla apart from conventional factors.

� Phase-I & Phase II Clinical studies of Shuddha Malla can be undertaken.

� With the collaboration of experimental pharmacology, various experimental

studies can be undertaken as Anticancer, Immunomodulator etc., to prove its

efficacy.

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LIMITATIONS

1. It was a time bound Research work.

2. The work was not monetarily sponsored.

3. Specific instrumentation and technological accreditation were taken from outside

accredited laboratories.

4. Specific laboratory assistance for results was taken from pathological labs.

5. The rat ADME profile for Arsenic may differ though it is pharmacologically similar

to that of humans for the need of extrapolation.

Apart from this all the efforts were made to give a prompt justice to the study with the

limited facilities. But the loopholes in the study may be excused by the learned.

.

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SUB-ACUTE TOXICITY Fig 55: HEART GROUP I GROUP III

28th Day -Normal 28th Day- Mild Infiltrations . GROUP V GROUP V

14th Day Mild Congestion 28th Day Oedematous Fibres GROUP V

28th Day Severe Oedema

Fig 56:BRAIN GROUP I GROUP III

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28th Day Normal 28th Day Mild Congestion GROUP V GROUP V

14th Day Congestion of Cells. 21st day Mild Degenerative Changes

GROUP V GROUPV

28th Day-Mild Congestion 28th Day -Mild Congestion & Infiltration of Few Mononuclear Cells.

Fig 57: LIVER GROUP I GROUP III

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28th Day -Normal 28th Day - Fatty Changes Degeneration Of Hepatocytes.

GROUP IV GROUP V

28th Day- Increased Kupffer Congestion & Haemorrhage Cells. (Modulatory Effect) 14th Day GROUP V GROUP V

21st Day-Fatty Changes 28th Day-Severe Haemorrhage Mild Haemorrhage Necrosed Hepatocytes. Swollen Hepatocytes.

154

GROUP V GROUP V

28th Day-Hepatocytes 28th Day-Hepatocyte Degeneration Separated,Congestion.

Fig 58: KIDNEY GROUP I GROUP III

28th Day - Normal 28th Day-Mild Congestion GROUP V GROUP V

21st Day-Severe Congestion 28th Day-Necrosis,Tubular Changes.

Fig 59: LUNG GROUP I GROUP V

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Mild Congestion & Oedema 28th Day-Mild Emphysema

Fig 60: SPLEEN GROUP I GROUP V

28th Day-Normal Mild Congestion & Haemorrhage 28th Day

Fig 61: GASTRIC MUCOSA GROUP V GROUP III

Keratinization with. Regenerative Changes More Slightly Proliferting Cells with More Basophilic Cells.

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ACUTE TOXICITY

Fig 48: HEART GROUP I GROUP IV

Normal Destruction of Cardiac Fibres GROUP VI GROUP VII

Congestion of Cells Severe Haemorrhage And destruction of Muscle Fibers.

Fig : 49 BRAIN GROUP I GROUP IV

Normal 12th Day Mild Congestion

GROUP IV GROUP VI

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14th Day- Mild Lesion Swollen Mild Lesion with Haemorrhage & Degenerating Neurons. 14th Day GROUP VII

Congestion, Mild Swollen Oligodendrites. GROUP VI GROUP VI I

Congestion, Infiltration of Haemorrhage & Infiltration Lymphocytes, Haemorrhage of Inflammatory Cells.

Fig 50: LIVER GROUP I GROUP IV

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14th Day- Normal Mild Fatty Infiltrations 12th Day

GROUP VI GROUP VI

Congestion of Cells-14th Day 14th Day Periportal Infiltration of Inflammatory Cells. GROUP IV GROUP VII

Severe Haemorrhage Hepatocytes Separated For with Swollen Hepatocytes accumulation Of Fluid GROUP VII GROUP VII

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Destruction of Hepatocytes Destruction Of Hepatocytes. Remnants of Epithelium.

Fig 51: KIDNEY GROUP I GROUP IV

Normal Mild Congestion & Degeneration. GROUP VI GROUP VII

Atrophic Glomeruli Congestion, Haemorrhage Tubular Changes Necrosis of Tubular Epithelium Degeneration, Necrosis. with Proteinacious Casts. GROUP VII GROUP VII

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Atrophic Glomeruli Swollen Tubules Haemorrhage Atrophic Glomeruli.

Fig 52: LUNGS GROUP I GROUP IV

Mild Congestion & Oedema Oedema,Haemorrhage Infiltration of Inflammatory Cells In Interstitia . GROUP VI GROUP VI

Severe Haemorrhage Thickening Of Alveolar Around The Vessel wall,Haemorrhage. GROUP VII

Periartiritis, Thickening of Alveolar Wall,Haemorrhage & Infiltration of Inflamatory Cells .

Fig 53: SPLEEN

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GROUP I GROUP IV

. Normal Mild Congestion GROUP VI I GROUP VII

Congestion & Haemorrhage Proliferation of Lymphoblasts around The Arteriole. Fig 54: GASTRIC MUCOSA GROUP I GROUP III

Normal Mild Lesion GROUP IV GROUP VI

162

Mild Keratinization Villi Showing Descumation Descumation of Krypts & Destruction or Gastric Glands. GROUP VII

Descumated Epithelium Due To Irritation.

PHARMACEUTICAL

163

Fig 3: Malla before Shodhana Fig 4: Karavellaka Fig 5: Karavellaka kalka

Fig 6: Extraction of Fig 7: Karavellaka swarasa Fig 8: Malla in Pottali Karavellaka juice

Fig 9: Dola yantra Swedana Fig 10: Boiling swarasa. Fig 11: After 5 hrs with lather after 2 hrs

Fig 12 : Pottali opened. Fig 13: Washing Malla pieces. Fig 14: Powdering Malla in after cooling Khalva. EXPERIMENTAL

164

Fig 15:Active rats in cage. Fig 16: Cages arranged. Fig 17: Feeding and water.

Fig:18 Dosage prepared. Fig 19: Weighing animals. Fig 20: Analytical balance.

Weighing the drug. Fig 21: Numbering the Fig 22: Tuberculin syringe rats.

Fig 23: Gavaging dose. Fig 24: Ether Chamber. Fig 25: Storage Vials.

165

Fig 26: Prepn of ether Fig 27: Blood drawing from chamber. retro-orbital plexus Fig 28: Capillary tube.

Fig 28: Blood in storage vial. Fig 29: Centrifuging machine. Fig 30:Semi-auto analyzer.

Fig 31: Dissection set. Fig 32: Diethyl Ether. Fig 33: Micropipettes with Serum

Fig 34: Clear Serum Fig 35: Reagent mixing. Fig 36: Analyzing serum .

Fig 36 Rehabilitating rat after. Fig 37: Blood clot after Fig 38: Separating anesthesia centrifuge Serum. Dissection of the rats after Sacrificing.

166

Fig 39:Rat in ether. Fig 40: Anaesthetized Rat. Fig 41: Decapitation of Rat. chamber

Fig 42: Dead Rat. Fig 43: Exposing Fascia. Fig 44: Excising the organs.

Fig 45: Dissection of Fig 46: Fixing organs in NBF. Fig 47: Ready

Brain. Containers

167

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166. Ibid,Chapter 3,31st pp.

167. Ibid ,chapter 3,32nd pp.

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171. Ibid,Chapter 4,49th pp.

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174. Ibid,Chapter 4,57th pp.

175. Ibid ,Chapter 5,64th pp.

176. Ibid ,Chapter 5,65th pp.

177. Ibid ,Chapter 5,69th pp.

178. A.Poole,G.B.Leslie,A Practical approach to toxicological investigations,First

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179. Ibid ,Chapter 5,74th pp.

180. Ibid ,Chapter 5,78th pp.

181. Ibid ,Chapter 6,87th pp.

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188. Ibid,Chapter 19,920th pp.

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