Certificate

I hereby certify that MR. ARUN M PRAJAPATI has completed his

Dissertation for Master of Pharmacy on the topic “Pharmacognostical and

Pharmacotechnical Evaluation of Kutaj Ghanvati” I further certify that

the work was carried out under my supervision and guidance at Department

of Quality Assurance, S. K. Patel College of Pharmaceutical Education and

Research, Ganpat Vidhyanagar, during the academic year 2005-2006. This

work is up to my satisfaction.

Date:

Place: Ganpat Vidhyanagar

Research Guide:

Dr. Rakesh K Patel

M.Pharm, Ph.D.

Ass Professor & Head,

Department of Pharmacognosy and Phytochemistry

SKPCPER, Ganpat Vidyanagar.

Head of department:

Dr. Paresh. U.Patel

M.Pharm, Ph.D.

Ass Professor & Head,

Department of Pharma chemistry

SKPCPER, Ganpat Vidyanagar.

Principal:

Dr. Prof. M. M. Patel

M. Pharm, Ph. D, F.I.C, L.L.B

S. K. Patel college of Pharmaceutical Education and Research

Research, Ganpat Vidyanagar – 382 711.

Declaration

I hereby declare that the topic entitled “Pharmacognostical and

Pharmacotechnical Evaluation of Kutaj Ghanvati” which is submitted to the

Hemchandracharya North Gujarat University, Patan, in partial fulfillment for

the award of degree of master of pharmacy in Pharmaceutical Quality

Assurance. The result of the work done by me in Quality Assurance

department under the guidance of Dr. Rakesh K Patel, Head, department of

Pharmacognosy and Phytochemistry.

I further declare that the results of this work have not been

previously submitted for any degree of fellowship.

Date: Arun. M. Prajapati

Place: Ganpat Vidhyanagar

Acknowledgement

S.K.P.C.P.E.R (M Pharm Dissertation) Arun M Prajapati I

ACKNOWLEDGEMENT

I take this opportunity to express my sincere gratitude towards my

respected sir and esteemed guide, Dr. Rakesh K Patel, Assistance

Professor and head, Department of Pharmacognosy and Phytochemistry of S.

K. Patel College of Pharmaceutical Education and Research, Kherva, It would

have never been possible for me to take this project to completion without

his guidance and support. I consider myself extremely fortunate to have had

a chance to work under his guidance.

I am extremely thankful to Dr.P.UPatel, Assistant Professor and

head, Department of Pharmaceutical chemistry for his valuable suggestions,

directions and selfless support throughout the investigation for providing

critical suggestions on my topic, and continuous guidance through out the

investigation.

I am grateful to our Incharge Principal Dr. Prof. N J Patel for

providing with the best facilities in the institute for completion of this work.

I also wish to thank whole heartily all the members of Pharmaceutical

Chemistry Department Dr.Mandev. B. Patel, Dr. Jignesh. R. Patel,

Satish A Patel, Bhavesh B Patel, Hiral Panchl, Shejal G Patel, Dipti

Patel for their continuous encouragement and valuable support to complete

my thesis work and my whole study period , and also for improving my

knowledge and helping me a lot.

I am thankful to Mr Kapil M Khambholja and Ms Nikunjana R Patel to give

me the great help with the knowledge of their field and For their guidance

Acknowledgement

S.K.P.C.P.E.R (M Pharm Dissertation) Arun M Prajapati II

I am extremely thankful to Dr. M M Patel, Principal, S. K. Patel

College of Pharmaceutical education and research, for giving me permition to

use laboratory facility of the college and continuous encouragement through

out my career as post graduate.

I am extremely thankful to MR Rakesh R Patel to give me a best

laboratory facility during my research work and always helpful through out

my research work. I also thank full to Madhuben A Patel for her help with

laboratory apparatus and chemical.

I am extremely thankful to Mr Sushilbhai Patel, Lab assistant, for

their moral moral support during my research work. Also Mr Jayeshbhai

Patel, who both provide me laboratory facility and every time to be helpful

in my practical work during my study. I also thank full to Mr Pravinbhai

Suthar, Mr Kanubhai Patel, and Mr Dipak Patel for their help throughout

my study.

I sincerely thank to Mr P I Patel, Librarian of S. K. Patel College of

Pharmaceutical Education and Research, Kherva, for providing me library

facilities and constant encouragement during my work.

I kindly thanks full to general manager, Cadila Pharmaceutical at Dholka to

provide me reference standards. I would like to express my special thanks to

the Advisor, Gujarat Council for Science and Technology, Gandhinagar,

for the financial support received in the form of Minor research project for

the work mentioned in this thesis, which was undertaken in the Dept of

Pharmacognosy of our college.

For help rendered by non-teaching staff especially Mr Dineshbhai

Patel (Store incharge) is sincerely acknowledged.

Acknowledgement

S.K.P.C.P.E.R (M Pharm Dissertation) Arun M Prajapati III

How can I forget them without whom all this work and my post

graduation can not be possible. I sincerely acknowledge the help rendered to

me by all my colleagues and friends paji (Jayesh), Kaushal, bipin, rishi,

Bhagat (Mitul), shital, Bhumi, Urvisha, and Bhavini during the course of

my work. A special mention of thanks to Dr Physco (Hitesh), Timir, Kirit

(Kito), Kirit (Bapu), Kalpo , Nandu, Pankajbhai, Sunil (Suno), Amatho,

Maheta, Ketan, and others for help rendered to me during the course of

project.

I must not forget guidance and help in every difficulty from my senior

Sanjay, Girish, Gayatri, Ritesh, Bhavik.

I would like to remember those who are always my well wisher and

friends, who help me throughout my study from junior KG to this level.

Ashvin, Dr saheb, Mitul, pintu, Alpesh (babo), Ila.

How can I forget those u give me company and great help through out

the Bachelor of Pharmacy. This work was not possible without their co-

operation and time to time discussion on the topics of study through out the

master of pharmacy. So I would like to thanks Bado (Nakul), Gondo (Nirav),

Kolad (Piyush), Vadi (Hitesh), Bathiyo (Viren), Thutho (Bhavesh), Kalu

(Kalpit),Maragho (darshan), Tejo (Tejash), modi (Mihir), Bhagat (Pratik),

Pappu (Piyush), Bavo (Rajendr), Lal (Sanjay), Asiyo (Ashish), Jado

(Pratik), Upalo (Upendr), Nikunj, Mihir,modi, hetal, bina, amit, priyanka,

bhumica, nili, ruta, vaishakhi, pankaj, bhavesh, bhagat hiren, dalvadi.

There is no need to mention name of my family member, I don’t have

to mention them and their work for me. This work and ultimately my self is

not possible without their care, love, guidance, co-operation, understanding

and trust on me. My Mama works behind my success and her loving care for

Acknowledgement

S.K.P.C.P.E.R (M Pharm Dissertation) Arun M Prajapati IV

me. I know they will not accept my thanks but I would like to thank my papa

specially to spend tots of money for him son. I extremely thank full to my

brother for taking me to this stage in life and always giving me guidance in

my every small mistake in life and study. I also thanks full to my brother’s

wife to take care for me and good support to maintain my routine college

time. It was the blessing of them that gave me courage to face the

challenges and made my path easier.

(Arun M Prajapati)

2005 - 2006.

List of Tables and Figures

S.K.P.C.P.E.R (M.Pharm Dissertation) I Arun M prajapati

List of Tables

Sr No Table Title Page No

1 Table 1.1 Survey of market formulation 2

2 Table 2.1 Uses of different parts of plants in different

system of medicine

18

3 Table 2.2 Chemical constituents of Kurchi 28

4 Table 4.1 Foreign matter, extractives and ash value of

Kurchi and Ativish

65

5 Table 4.2 Total alkaloids of Kurchi and Ativish 66

6 Table 4.3 Assay of conessine in different samples of

Kurchi bark by HPTLC

67

7 Table 4.4 Regression parameters for the analysis of

conessine by HPTLC

68

8 Table 4.5 Data of recovery study of conessine by HPTLC 69

9 Table 4.6 Method Precision data of analysis of conessine

by HPTLC

69

10 Table 4.7 Intra-day precision data of analysis of conessine

by HPTLC

70

11 Table 4.8 Inter-day precision data of analysis of conessine

by HPTLC

70

12 Table 4.9 Summary of validation parameters of conessine

by HPTLC

72


S.K.P.C.P.E.R (M.Pharm Dissertation) II Arun M prajapati

13 Table 4.10 Assay of atisine in different samples of ativish

by HPTLC

74

14 Table 4.11 Parameters of regression for the analysis of

atisine by HPTLC

75

15 Table 4.12 Data of recovery study of analysis of atisine by

HPTLC

76

16 Table 4.13 Method Precision data for the analysis of atisine

by HPTLC

76

17 Table 4.14 Intra-day precision data for analysis of atisine by

HPTLC

77

18 Table 4.15 Inter-day precision data for analysis of atisine by

HPTLC

77

19 Table 4.16 Summary of validation parameter of atisine by

HPTLC

79

20 Table 5.1 Ash values of samples of Kutaj Ghanvati 91

21 Table 5.2 Assay of Kutaj Ghanvati for the total alkaloids 91

22 Table 5.3 Assay of conessine and atisine in different

samples of Kurchi bark by HPTLC

93

23 Table 5.4 Regression Parameter for analysis of conessine

and Atisine by HPTLC

94

24 Table 5.5 Data of recovery study of conessine and atisinet

in Kutaj Ghanvati by HPTLC

95

25 Table 5.6 Method Precision data of analysis of conessineb

and atisine by HPTLC

96

26 Table 5.7 Intra-day precision data of analysis of conessine


97

27 Table 5.8 Inter-day precision data of analysis of conessine


97


S.K.P.C.P.E.R (M.Pharm Dissertation) III Arun M prajapati

28 Table 5.9 Summary of validation parameters of conessine


99

29 Table 5.10 Friability, Disintegration and Crushing strength

of Kutaj Ghanvati

101

30 Table 5.11 % of total alkaloids of Kutaj Ghanvati released

after 2 hour.

101


S.K.P.C.P.E.R (M Pharm Dissertation) I Arun M Prajapati

List of Figures

Sr No Figure Title Page No

1 Figure 1.1 Transverse section of Kurchi 4

2 Figure 1.2 Powder characters of Kurchi 5

3 Figure 1.3 Structure of conessine 6

4 Figure 1.4 Trasverse section of Ativish 9

5 Figure 1.5 Powder characters of Ativish 10

6 Figure 1.6 Structure of atisine 11

7 Figure 4.1 Powder characters of Kurchi bark powder 64

8 Figure 4.2 Powder characters of Ativish root powder 64

9 Figure 4.3 Photograph of a TLC plate showing separation of

conessine

67

10 Figure 4.4 Calibration curve of conessine by HPTLC

method.

68

11 Figure 4.5 Inter-day precision data of analysis of conessine

by HPTLC method

71

12 Figure 4.6 Chromatogram of Kurchi bark 71

13 Figure 4.7 UV Spectra of Atisine show maximum absorption

at 274 nm

73

14 Figure 4.8 Photograph of the plate showing spots of atisine

from standard solutions

73

15 Figure 4.9 Calibration curve of atisine by HPTLC method at

520 nm

75

16 Figure 4.10 Calibration curve for analysis of Atisine by

HPTLC method at 274 nm.

75

17 Figure 4.11 Chromatogram of atisine in Ativish 78

18 Figure 4.12 Chromatogram of atisine standard at 274 nm 78


S.K.P.C.P.E.R (M Pharm Dissertation) II Arun M Prajapati

19 Figure 4.13 Chromatogram of atisine standard at 520 nm 79

20 Figure 5.1 Microscopic characters of ativish in Kutaj

Ghanvati

90

21 Figure 5.2 Photograph of a plate containing chromatograms

obtained from standard solutions of conessine and

atisine.

92

22 Figure 5.3

Photograph of a TLC plate containing

chromatograms obtained from formulation of

Conessine and atisine.

93

23 Figure 5.4 Calibration curve of analysis of atisine by HPTLC

method

94

24 Figure 5.5 Calibration curve of analysis of conessine by

HPTLC method

95

25 Figure 5.6 Chromatogram of conessine and atisine

formulation

100

26 Figure 5.7 Chromatogram of conessine and atisine standard 100

Abbreviation

S.K.P.C.P.E.R (M Pharm Dissertation) I Arun M Prajapati

Abbreviation

Gm Gram

Rs Rupees

TS Transverse section

p Prisms:

ck Cork;

ct Cortex;

mr Medullary ray

p Prisms;

Pf Pericyclic fibers;

Ph Phloem;

stc Stone cells.

C' Outer zone of cortex;

En Endodermis;

C' Broader inner zone of cortex;

Sl Stone cells;

Cam Cambium;

T Trachea of xylem

Si Sieve tubes of phloem of bundle;

M Pith.

WHO World health organization

CCD Continuous capture device

IP Indian Pharmacopoeia

HPTLC High performance liquid chromatography

TLC Thin layer chromatography

ICH International conference on harmonization

nm Neno meter

µl Micro litre

µg Micro gram

% w/w Percentage Weight/Weight

Abbreviation

S.K.P.C.P.E.R (M Pharm Dissertation) II Arun M Prajapati

% v/v Percentage Volume/Volume

UV Ultra violate

S.D Standard deviation

RSD (% CV) Relative standard deviation

LOD Limit of detection

LOQ Limit of Quantitation

Table of contents

S.K.P.C.P.E.R (M Pharma Dissertation) Arun M Prajapati I

Table of Contents

Chapter Title Page No

1 Introduction 1-15

1.1 Ghanvati

1.2 Kutaj Ghanvati

1.3 Kurchi

1.4 Ativish

1.5 References

2 Literature review 26-49

2.1 Kutaj Ghanvati

2.2 Kurchi

2.3 Ativish

2.4 References

3 Aim of the work 50-51

4 Evaluation of raw materials 52-80

4.1 Introduction

4.2 Experimental

4.2.1 Pharmacognostic and physicochemical evaluation of

Kurchi

4.2.2 Determination of total alkaloids of Kurchi

4.2.3 Estimation of total alkaloids of Ativish

4.2.4 Estimation of conessine in Kurchi by HPTLC

4.2.5 Estimation of atisine in Ativish by HPTLC

4.3 Results and Discussion

4.3.1 Pharmacognostic and physicochemical evaluation of

Table of contents

S.K.P.C.P.E.R (M Pharma Dissertation) Arun M Prajapati II

Ativish

4.3.2 Total alkaloids of Kurchi and Ativish

4.3.3 Estimation of conessine in Kurchi by HPTLC

4.3.4 Estimation of atisine in Ativish by HPTLC

4.4 References

5 Evaluation of Kutaj Ghanvati

80-102

5.1 Introduction

5.2 Experimental

5.2.1 Preparation of Kutaj Ghanvati

5.2.2 Pharmacognostic and physicochemical evaluation of Kutaj

Ghanvati

5.2.3 Determination of total alkaloids of Kurchi in Kutaj

Ghanvati

5.2.4 Estimation of conessine and atisine in kutaj Ghanvati by

HPTLC

5.2.5 Evaluation of tablet parameter of Kutaj Ghanvati


5.3.1 Pharmacognostic and physicochemical evaluation of Kutaj

Ghanvati

5.3.2 Total alkaloids of Kutaj Ghanvati

5.3.3 Simultaneous estimation of conessine ant atisine in Kutaj

Ghanvati by HPTLC

5.3.4 Evaluation of tablet parameters of Kutaj Ghanvati

5.4 References

6 Conclusion

103-104

Chapter-1 Introduction

S.K.P.C.P.E.R (M. Pharm Dissertation) Arun. M. Prajapati 1

Chapter 1

Introduction

1.1 Ghanvati (Vati)

Vati is presented as tablet or pill. These are made up of various drugs of minerals,

animals and vegetable origin1.

1.1.1 Method of preparation of Ghanvati

The vegetable drugs are dried and made into fine powders, separately. The minerals

are reduced to bhasma or sindura, unless otherwise mentioned in case where parada

and ganataka are mentioned, kayagali is made first and other drugs are added

according to the formula. These are put into the khalva and ground to a soft paste with

the prescribed liquids, when more than one ground is mentioned for grinding. They

are used in a succession. When the mass is properly ground and is in a condition to be

made into pills. Sugandha like kasturi, kurpura if mentioned are added and ground

again.

The criteria to determine the final stage before making pills is that it should not stick

to the fingers when rolled. Pills may be dried in shape or under sun in accordance with

the textual directions in case of where sugar or jeggery (guda) is mentioned paka of

these should be made in low fire and remove from the oven. The powder of the

ingredients are added to the paka and briskly mixed. When warm, vatikas should be

rolled and dried in shape2.

1.1.2 Preservation and characteristics

Vati made of vegetable origin kept in airtight containers can be made of minerals can

be used for an indefinite period. Vati should not loss their marginal color, smell, taste

an d form, when sugar is added then pills are stored away from mixture2.



1.2 Kutaj Ghanvati

Kutaj Ghanvati is famous Ayurvedic formulation, which is given in the Ayurvedic

formulary and used in various disorders such as dysentery, diarrhoea and other

aliments. In Ayurveda ghanvati means pills or tablets. Kutaj ghanvati contains two

drugs- Kurchi and Ativish. According to ayurveda water extracts of Kurchi bark

powder and dried powder of the root of Ativish is incorporated and prepared

according to the procedure described under the general method of preparation of

Ghanvati.

Indication

Kutaj ghanvati is used in dysentery and diarrhoea2.

Dose

2-3 Tablets per day

Market preparations

We made survey of the local market and the information collected which are

mentioned in Table1.1

Table 1.1 Survey of market formulation

Company

name

(Pharmacy)

Preparation

name

Weight of tablet

(gm)

Dose

(tablet/day)

Prise

(Rs)

Zandu

Dabur

Bhuvaneshvari

Nimbark

Shankar

Vishvamitri

Narnarayan

Unja

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

Kutaj Ghanvati

390

750-1250

200-300

400

540

500-1000

390

240-390

2-4

2-4

1-2

2-3

2-4

2-4

1-2

1-2

33

40

22

21

24

27

32

36



1.3 Kurchi

Botanical source

Drug consists of dried stem bark of Holarrhena antidysentrica (Roth)3.

Family: Apocynaceae

Geographical source

It is distributed through out India, especially in the west forest and tropical Himalayas,

up to an altitude of 1,200m.

Vernacular name

Beng- Kurchi

Eng- Conessi, Kurchee

Guj- Kado

Hind- Kuraiya

Kan- Kodasige

Mar- Kudda

Mal- Kodagapal

Punj- Kewar, Kura

Tam- Veppalar

Tel- Kaka-kodise

Macroscopic description

Recurved pieces of bark of varying sizes and thickness, buff to reddish brown with

numerous prominent circular or transversely elongated horizontally placed lenticels,

longitudinal wrinkles with a rough and brownish inner surface; odourless, taste bitter3.

Microscopic description

TS of stem bark shows periderm, a wide cortex and secondary phloem (Figure 1.1).

Periderm consists of thin- walled and somewhat rectangular cork cells. 2 to 3 layers of

phellogen and parenchymatous cells of phelloderm containing prism of calcium



oxalate crystals and a few starch grains. Cortex is interspersed with groups of lignified

pitted stone cells of different sizes and shapes. Prism of calcium oxalate crystals is

present in parenchyma and in some stone cells, which is a characteristic feature of the

stem bark. Occasional groups of non-lignified pericyclic fibers are seen in cortex.

Continuous bands of stone cells are present in phloem region. Medullary rays are bi or

tri-seriate. Prisms of calcium oxalate crystals and starch are abundantly present in

phloem parenchyma. Absence of phloem fiber is a conspicuous feature of this work4

(Figure 1.1).

Figure 1.1 Transverse section of Holarrhena antidysentrica bark. p. prisms: ck.

cork; ct. cortex; mr. medullary ray: p, prisms; pf, pericyclic fibers; ph, phloem;

stc, stone cells.



Powder characters

Light brown, taste bitter; thin-walled cork cells, groups of stone cells of different sizes

and shapes. Prisms of calcium oxalate crystals in parenchymatous cells and in stone

cells and also scattered all over (Figure 1.2)

Figure 1.2 Microscopic character of Holarrhena antidysentrica bark powder



1. Stone cells (st) and sclereids (scl).

2. Prismatic crystals of calcium oxalate.

3. Starch grains. ,

4. Cork in surface view.

5. Parenchyma of cortex filled with starch grains and prismatic crystals of

6. Calcium oxalate.

7. Phloem parenchyma filled with starch grains & prism crystals of ca.oxalate.

Chemical constituents

Major

Total alkaloids ~4.0 percent5; bioactive steroidal alkaloid Conessine

6 0.4 % (Figure

1.3) Kurchisine, Conkurchine, Holarrhine7.

Figure 1.3 Structure of conessine

Other

Steroidal alkaloid kurchiline, Kurchiphyllamine, Conessimine, hollarhimine,

norconessine, Conessidine, conamine, Conarrhimine, Isoconessimine, conimine,

Lettocine, conkurchinine, holrrhesmine, Kurchessine, holanamine, Holarrhidine,

Holantosine A, B, C, D, E, Holarosine A, Trimethyl Conkurchine7; Regholarrhenine

A, B, C, D, E, F8, 9

, Holarrifine10

: 5.20(29)-lupadien-3-ß-ol and sitosta-511

.

Pharmacopoeial specification12

Total alkaloids: Not less than 2%

Foreign matter: Not more than 2%

Total ash: Not more than 7.5%

Acid insoluble ash: Not more than 8%



Ethanol-soluble extractive: Not less than 21%

Water-soluble extractive: Not less than 27%

Loss on drying: Not less than 8%.

Adulterants/Substitutes

Stem bark of H. antidysentrica is often adultrated with Wrightia tinctoria and W.

tomentosa, which can be distinguished by the morphological and microscopic

features13-16

.

Pharmacology

H. antidysentrica is effective in acute and chronic amoebic dysentery. Various fraction

of the drug showed promising activity against amoebiasis in rats and hamsters17

.

Conkurchine hydrochloride at higher doses decreases heart rate of frog lowered

dowered dog blood pressure and dilated rat blood vessels18

. The drug also possesses

antibacterial activity19

.

Therapeutic category: Antidysenteric20-21

.

Safety aspects: Hypertensive effect was reported22

.

Dosage

Stem bark powder: 3 to 6 g20

Decoction: 20 to 30 g21.



1.4 Ativish

Botanical source

Drug consists of dried root of Aconitum heterophyllum (Roth) .

Family: Ranunculaceae

Geographical source

A large, evergreen terr, indigenous to the evergreen forests of the Western Ghats,

altitude, 450-1200 m, cultivated in plains, almost throughout India.

Vernacular name

Ben - Kanthal, Kantghel, Kathal

Eng- Jackfruit, Jacktree, Indian Jack-tree

Guj- phanas, Manphansa

Hind- Kanthal, Kathal, Panasa, Katahara

Kan- Halasu, Hebhalasu

Mal- Chakka (fruit), Pilavu (tree), Pilva

Mar- Phanas

Ori- Ichodopholo, Katokola, Ponoso

Tam- Murasabalam, Pala, Pila, PIlapalam,

Tel- Panasa, Verupansa

Macroscopic description

The tuberous roots occur either singly or in clusters of 2 or 3. When the younger

smoother root is connected with the older wrinkled root by means of side branch. Each

root is somewhat conical or fusiform, from 1to 3.5 cm. In width at the crown;

externally dark brown or grayish-brown, smooth or longitudinally wrinkle, the upper

end with a bud or remains of bud scales (young daughter roots) or stem scars r basal

portions of stems scars or short rootlets; fracture short, horny or mealy; internally, old

roots are brownish while young roots are whitish, exhibiting a 5 to8 angle cambium

with a small fibrovascular bundle in each angle23

.



Microscopic description

TS of root, cut near the middle of the tuberous root, exhibit the following structure 1)

Acork region of one or more layers of blakish or brownish cells. 2) A broad cortex of

two region, viz: an outer narrower an inner broader zone. The narrower zone consists

of from 8 to 15 layers of parenchyma, with numerous stone cells. Separating this zone

with single layer of endodermis of tangentially elongated endodermal cells. The

boarder region consists of starch containing parenchyma cells, with few fibrovascular

bundles. Stone cells may also occur in this zone beneath the endodermis. 3) A 5 to 8

angled cambium, more or less star-shaped, within the angles of which, and frequently

scattered along the entire cambial line occur collateral fibrovascular bundles. 4) A

broad 5 to 8rayed pith composed of parenchyma cells. The parenchyma cells of both

cortex and piyh contain numerous single or 2 to 5compound starch grains as well as

active principles. Most of the active principles are localized in the cortex just outside

of the angles of thw cambium23

(Figure 1.4).

FIGURE 1.4. Transverse section of Aconitum Heterophyllus root. Transverse

Section of root K, cork; C', outer zone of cortex; En, Endodermis; C', broader inner



zone of cortex; Sl, stone cells; Cam, cambium; T, trachea of xylem and Si; sieve tubes

of phloem of bundle; M, pith.

Powder characters

Grayish-brown, starch grains abundant, spherical or plano-convex, single or 2-5

compound, the individual grains from 3 to 20 in diameter and frequently showing a

central cleft hilum; stone cells strongly lignified or elongated to fibers up to 400 long

with porous walls up to 25 thick; fragments of yellowish-brown cork; fragment of

parenchyma filled with starch; sclerenchyma fibers from stems few, very longwidth

lignified walls about 5-6 in thickness and showing oblique or transverse slit-like

pores; tracheae for the most part with simplepores but spiral, reticulate and border

pored tracheae also present23

(Figure 1.5).

1 2 3

Figure 1.5 Microscopic characters of ativish root powder

1 Xylem vessel

2 Starch grain

3 Cork cell

Chemical constituents

Major

The root contains non-toxic, amorphous alkaloids, atisine (0.4%)24

(Figure 1.6),

atisenol dehydroatisine, heteratisine and hetisine25

. It also contains aconitic acid,

tannic acid, pectin and starch. Total alkaloids present in plant is not more than ~5.0

percent.



Figure 1.6 Structure of Atisine

Other

Morin (3, 5, 7, 2’, 4’- pentahydroxyflavone)26-28

, Dihydromorin, Artoflavanone

(5-Hydroxy- 6-C—prenyl-7, 3’, 4’, 5’- tetramethoxy flavanone)29-30

,

Oxydihydrocarpesin31

, 9, 19-Cyclolanost-3-one-24, 25-diol (24R) etc, are found in

root32

.

Pharmacopoeial specification33

Total alkaloids: Not more than 5%

Foreign matter: Not more than 2%

Total ash: Not more than 5%

Acid insoluble ash: Not more than 1%

Ethanol-soluble extractive: Not less than 17%

Water-soluble extractive: Not less than 23%

Loss on drying: Not less than 6%

Adulterants/Substitutes

Two Japanese aconite roots one is Aconitum fischeri, which contain jesaconitine. And

other is A uncinalum and japonicum, which contain japaconitine. Root of A

chasmanthum, A nepelus, A ferox other few Aconitum species verities34

.

Pharmacology



The unripe fruit is acrid, astringent, carminative, and tonic. The ripe fruit is

demulcent, nutritive, laxative, cooling, fattening, and useful in biliousness, the seeds

are diuretic. The leaves are used in skin diseases. Ash of the leaves is useful in healing

ulcer. The root if said to be useful in skin disease, asthama, and diarrhea. Juice of the

plant is applied to glandular swelling and abcesses to promote suppuration. It is also

used for snakebite23

.

Therapeutic category: Antimicrobial, Anthelmintic35-38

.

Safety aspects: poisonous38-39

.

Dosage

Small dose of 0.05mg per day40.



1.5 References

1. Ayurvedic formulary, part-I; 153.

2. Siddha Yog Sangraha, 8th

edition, 1984; 24.

3. Satyavati G V, Gupta A K, Tandon N, editor. Medicinal plants of India, Vol. II.

New Delhi: council of medical research; 1987; 41-49.

4. Iyengar M A, Nayak S G K. Anatomy of crude drugs. 8th

ed. Manipal: prof.

Iyengar M A; 2001; 37-38.

5. Bhutani K K, Raj S, Gupta D K, Kumar S. Atal C K. Kaut M K. Profile of Kurchi

in India. Indian drugs 1984; 21:212-216.

6. Siddiqui S. the alkaloids of Holarrhena antidysentrica. IV. The occurrence of

further new two bases in the bark of Indian Holarrhena and their relation to

Conessine and holarrhemine. Proc Indian Acad Sci 1936; 3A: 249-256.

7. Chaturvedi G N, Singh K P, Gupta J P, phytochemistry and pharmacology of

Holarrhena antidysentrica WALL. (Kutaj). Indian Med Gaz 1981; 115: 179-972.

8. Bhutani K K, Ali M, Kapoor S, Soodan SR, Kumar D. steroidal alkaloid from the

bark Holarrhena antidysentrica. Phytochemistry 1990; 29: 969-972.

9. Bhutani K K, Ali M, Sharma S R, Vaid RM, Gupta DK. Three new alkaloids from

the bark of Holarrhena antidysentrica. Phytochemistry 1988; 27: 925-928.

10. Siddiqui S, Shamsuddin B A. Isolation and structure of holarrifine, new alkaloids

from the bark of Holarrhena antidysentrica Linn Pak J Sci Ind Res 1989; 32: 1-3.

11. Narayanan C R, Naik D G. A new triterpine and steroid from Indian Kurchi bark.

Indian J Chem 1981; 20B: 62-63.

12. Indian Pharmacopoeia, 1955; 358.

13. Prasad S, Kaul P N, Pharmacognostical study of Holarrhena antidysentrica and

Wrightia tomentosa barks. Indian J Pharm 1956; 18: 423-4445.

14. Atal C K, Sethi P D. Wrightia tinctoria bark an adulterant of Kurchi bark. J Oharm

Pharmacol 1962; 14: 41-45.

15. Datta S C, Bal S N. Pharmacognostical studies of Holarrhena antidysentrica.

Indian J Pharm 1945; 7: 113-116.

16. Gopal V, Chauhan M G. Holarrhena antidysentrica- a review in: Handa S S, Kaul

M K, and editors. Supplement to Cultivation & Utilization Of Medicinal Plants.



Jammu-Tawi: Regional Research Laboratory. Council Of Scientific And Industrial

Research 1996; p. 223-245.

17. Datta N K. Iyer S N. Antiamoebic value of berberine and Kurchi alkaloids .J

Indian Med Assoc 1968; 50: 349-352.

18. Shankar J. Neogi N C, Basu N K. Pharmacological studies on conkurchine

alkaloids. The Proc Rajasthan Acad Sci 1961; 8:94-97

19. Chakraborty A. Brantner A H. Antibacterial steroid alkaloids from the stem bark

of Holarrhena pubescens. J Ethnopharmacol 1999; 68: 339-344.

20. Sharma P V. Classical uses of Medicinal Plants .1st Edi.Varansi: Chaukhambha

Viswabharati (Oriental Publishers & Distributors); 1996; p.101-103

21. The Ayurvedic Pharmacopeia Of India, Part-1 vol. 1. 1st Ed. New Delhi:

Government of India, Ministry of health &Family Welfare, Dept Of Health;

1989:p. 78-79

22. Chturvedi G N, Singh K P. Side effects of Traditional indogenous drug- kutaj

(Holarrhena antidysentrica). Indian J Physiol Oharmacol 1983:27:255-256.

23. Wealth of India, Raw material-I, A., revised edition, New Delhi1985; 61-62.

24. Alexander L, James E C T., J. Chem. Soc., 1937, 1640-1643.

25. Walter A J, Lyman C C., The isolation of two new alkaloids from aconitum

heterophyllum, heteratisine and hetisine.1942; 605-609.

26. Perkin and Cope 1895. J Chem Soc 67, 937. Quoted in Radhakrishnan, P.V. and

Rama Rao, A.V. 1966. Indian J Chem 4, 406-412.

27. Chakravarty, G. and Seshadri, T.R. Structure of Cynomaclurin, a component of

jack wood. Tetrahedron Lett No 18, 1962; 787-794.

28. Dave, K.G. and Venkatraman. K. The coloring matters of the woods of Artocarpus

integrifolia. Part I-Artocarpin. J Sci Ind Res 15B, 1956; 183-190.

29. Dave, K.G., Mani, R. and Venkatraman, K. The coloring matters of the wood of

Artocarpus integrifolia: Part III - Constitution of Artocarpin and synthesis of

tetrahydroartocarpin dimethyl ether. J Sci Ind Res20B, 1961; 112-120.

30. Dave, K.G., Telang, S.A. and Venkatraman, K. Flavonoid pigments of .the

heartwood of Artocarpus integrifolia. Tetrahedron Lett No 1, 1962; 9-14.



31. Parthasarathy, P.C., Radhakrishnan, P.V., Rathi S.S. and Venkatraman, K.

Coloring matters of the wood of Artocarpus heterophyllus: Part V.

Cycloartocarpesin and oxydihydroartocarpesin, two new flavones. Indian J Chem

7, 1969; 101-102.

32. Barik, B.R., Bhaumik, T, Dey, A.K. and Kundu, A.B. Triterpenoids from

Artocarpus heterophyllus. Phytochemistry 35, 1994; 1001-1004.


34. Natural drugs morphological and taxonomic consideration, 2nd

edition, Herder

Wilkinson yongken- New Delhi 2003; 206-215.

35. Valsaraj, R., Pushpangadan, P., Smitt, U.W., Adserson, A. and Nyman, U.

Antimicrobial screening of selected medicinal plants from India. J

Ethnopharmacol 58, 1997; 75-83.

36. Sharma, N. Fungitoxic properties of plant latex against some post harvest diseases.

Bioved 5, 1994; 81-84.

37. Siddiqui, M.A., Haseeb, A. and Alam, M.M. Evaluation-of nematicidal properties

in some latex bearing plants. Indian J Nematol 17, 1987; 99-102.

38. Siddiqui, M.A., Haseeb, A. and Alam, M.M. Control of plant-parasitic nematodes

by soil amendments with latex bearing plants. Indian J Nematol 22, 1992; 25-28.

39. Sharma, W. and Trivedi, P.C. Nematicidal and nematostatic response of aqueous

extract of certain plants of semi arid niche. Curr Nematol 6, 1995; 45-53.

40. Nath, M.C. and Sengupta, T. N.Sex-hormone activities of artostenone derivatives.

Part I. Action of artostenone on sexually immature male rats. Indian J Med Res 27,

1939; 171-179.

Chapter-2 Literature review

SKPCPER (M.Pharm Dissertation) Arun M Prajapati 16

Chapter 2

Literature review

2.1 Kutaj ghanvati

Bhavsar et al., 20041,2

, studied the standardization of Kutaj ghanvati and reported

total alkaloids in the Kutaj ghanvati as per the method of Indian Pharmacopoeia. They

found out that extraction with methanol:chloroform:ammonia gave better results of

total alkaloids in ativish than the method of IP. They have shown the separation of the

alkaloids in TLC but not quantified. Kudalkar et al., 19823

done standardization of

kutaj ghanvati for total alkaloids of Kutaj Ghanvati.



2.2 Kurchi

Holarrhena R. Br (Apocynaceae) is a genus of trees or shrubs distributed throughout

the tropical and subtropical region of the world. About eight species of this genus are

known but only “ Holarrhena antidysentrica (linn) Wall, Synonym Holarrhena

pubescenta (Buch-Ham) Wall grows in India (Trease and Evans et al., 1989)4. In

Sanskrit it is commonly known as kutaja. The different parts of the plant were used

since adequity in the indigenous system of medicine but the stem bark and the seeds

were more extensively employed as antidiarrhoeal and anthelmintics drugs. Seeds are

sold as name “Indrayava” while the bark under the name of

“Kurchi”,”Conessi”,”Tellicherry” or ”Koora” is confusing because some of its

commercial adultrants like Wrightia tinctoria and Wrightia tomentosa are also

labelled as “Koora” (Kaul and Atal et al., 1983)5.

Kirtikar and Basu et al., 19336 mentioned the use of the parts of the expect flower in

cases of snakebite and scorpion sting. Evidences of chronic cases of dysentery which

could not be cured by European medical treatment (Nadkarni et al., 1955)7

and

substitution of kurchi bark in place of emetine has also been suggested (Nandi and

Majumdar et al., 1979)8.

Jain and Terafder et al., 19709 have mentioned the use of this plant by the various

Indian tribals in cases of number of ailments like anaemia, epilepsy, obstetric,

condition, spermatorrhoea, haematuria, constipation, stomachache, and cholera and in

dog bites. Sharma et al., 197910

recommended the seeds in case of jaundice. Gopal

and Chauhan et al., 199311

have documented the use of kurchi seeds in the diabetes

mellitus.

In Ayurveda, the plant has been extensively used in the treatment of various bleeding

disorders like diarrhoea, dysentery, piles, abortions, invisible hemorrhoids etc.

Sushruta has advocated the use of flower in prameha (diabetes). Kaul and Atal et al.,

198312

have summarized the use of various parts of the plant in the different systems

of medicines as in Table 2.1.



Table 2.1 Uses of different parts of plants in different system of medicine

Sr.

no.

Part used System of medicine Uses

1 Stem bark Ayurveda Anthelmi9ntic, stomachic astringent,

in cases of diarrhoea, fever, piles,

leprosy, thirst, skin diseases, diseases

of spleen, dropsy, and biliousness.

Yunani Used against headache, strengthens the

gums, reduces in inflammation and

excessive menstrual flow.

Portuguese Used as a plaster in rheumatism, as a

hot decoction in toothache and bowel

infections.

British materia

medica

Antidysentric.

2 Seed Ayurveda Cooling, appetizer, carminative,

astringent, anthelmintics: in case of

leprosy, burning sensation, dysentery,

skin disease, biliousness: bleeding

piles, fatigue and hallucinations.

Yunani Carminative, astringent lithon-triptic

and aphrodiastic, used against chronic

chest infections, pessaries made with

honey and honey and saffron are

supposed to favour conception.

3 Leaves Yunani Astringent, galactagogue, tonic,

aphrodiastic, mitigates pain in

muscles, cools the brain. Useful in

cases of chronic bronchitis, lumbago,

urinary discharge, boils, ulcers,

wounds, burns, regulating

menstruation and for fumigating the

mother and child after delivery.

4 Flowers Ayurveda Appetizer, anthelmintics,

antihepatotoxic, antidiarrhoeal, and in

disease of blood and leucoderma.

Besides the system its use in homeopathic system of medicine has also been

mentioned by (Nandi and Majmudar et al., 1979)13

. Singh and Chaturvedi et al.,

198214

have mentioned its use as a single drug therapy or in combination with other

drug in almost all types dosage forms, and have cited out about 20 formulation

containing the bark or seeds of kurchi as one of the major ingredient.



Chopra et al., 195615

has also mentioned the commercial use of the leaves for bidi

making and the Wealth of India, 195916

mentioned the use of wood for making small

articles such as combs, boxes, cups, ploughs, mathematical instruments and furniture.

Prasad and Kaul et al., 195617

have described in detail the Pharmacognosy of kurchi

bark and its adultrant Wrightia tomentosa while Atal and Sethi et al., 196218

have

described the Pharmacognosy of its another adultrant Wrghtia tinctoria. Khan et al.,

198719

studied the comparative morphological and microscopical characters of the

seeds of H. Antidysentrica and one of its adultrant W.tinctoria available in the market

under the name of “Sweet indrajava”.

Alkaloids have been reported in the bark, leaf, and seed parts of the plant. Bark is rich

in alkaloids which are located in the phloem not in the periderm Dutta and Bal et al.,

194520

the alkaloid content of the bark contains maximum amount of alkaloids girth of

the plant. Prasad and Kaul et al., 195721

Eight years old stem contain maximum

amount of alkaloids (3-4%) and hence are used in the commercial scale production of

Holarrhena alkaloids and hydrochlorides and Holarrhena bismuth iodide. Bhutani et

al., 198422

have reported the alkaloid content of the (0.6-3.90%), leaf (0.6-1.4%) and

seed (0.30-0.91%) of widely growing trees from different region of India, the highest

(4.72%) being i9n the stem bark collected from Gujarat state. The commercial content

of the commercial samples of bark (1-2.3%) and seed (0.7%) from the different region

of India where also reported by them. the adultrant s of kurchi bark W. tomentosa

contain 1.55% (Jayswal et al., 1977)23

and 0.4% W.tinctoria (Bhutani et al., 1984)24

(Dutta et al., 1950)25

studied the plant content seasonally. The highest found after rain

and in the month of November (stem: 3.89%, root: 3.76%) and December (stem:

3.78%; root: 3.8%) while the leaf contain highest amount in June (1.56%). The

alkaloid content of stem (1%) remains constant throughout the year. considerable

work has been carried on the chemistry and biology activity of kurchi which has been

reviewed by several authors like; (Roy and Mukherji et al., 1958)26

, (Bhandari and

Mukherji et al., 1959)27

, (Gunnar et al., 1968)28

and (Chaturvedi et al., 1980-

1981)29

. A bibliography on kurchi has also been published anonymous.

Alkaloids isolated from the kurchi are listed below. Conessine (Haines et al., 1858)30

,

Kurchicine, Kurchine (Ghosh et al., 1928)31

, Nor Conessine (Robert et al., 1932)32

,



Conessimine and Holarrhine and Holarrhimine (Siddqi et al., 1932)33

, Conessidine

and Conkurchine and Curchinine (Bertho et al., 1933)34

, Conimine and

Isoconessimine (Siddiqui et al., 1934)35

, Lettocine (Peacock et al., 1935)36

,

Conamine and Conarrhimine (Siddiqui et al., 1934)37

, Holarrhensimine (Tschesche et

al., 1954)38

, Trimethyl Conkurchine (Tschesche and Roy et al., 1954)39

, Holarrhidine

(Labler et al., 1957)40

, Kurchamine and Kurchessine and Tetra methyl Holarrhimine

and (3)-N-Methyl Holarrhimine-2HCL and (20)N-Methyl Holarrhlmine (Tschesche et

al., 1958)41

, Kurchimine and Kurcholessine (Tschesche and Peter et al., 1962)42

,

Dihydroconcurressine and concurressine and Epihetroconessine (Labler and Sorm et

al., 1963)43

, Dihydroisoconissimine and 3a-aminoconan-5-ene and 7a-

hydroxyconessine and Holonamine (Cerny et al., 1964)44

, 7a-hydroxyconessine and

Holonemine (Tschesche and Ockenfels et al., 1964)45

, Kurchiphyllamine and

Kurchiphylline and Kurchilline and Kurchaline and Holantosines A,B and

Holantosine C, D (Janot et al., 1966)46

, Holarosine-A (Qui et al., 1971)47

,

Holantosine E, F and Holarosine-B (Goutarel Robert et al., 1972)48

, Holacetine (Rej

et al., 1971)49

, Holarricine and Holacine and Holacimine (Siddiqui et al., 1981-

1982)50

, Regholarrhenine A,B,C (Bhutani et al., 1988)51

, Holarrifine (Siddiqui et al.,

1989)52

, Regholarrhenine D,E,F (Bhutani et al., 1989)53

.

Siddiqui et al., 193454

studied the action of cynogen bromide on Conessine and its N-

demethylation to Isoconessimine and Conimine. Irani et al., 194655

isolated 1.4% of

glyco alkaloid and galactose as one of its hydrolysis product. Alfred Bertho et al.,

195356

substantiated the structure of Conessine and Conkurchine. Haworth et al.,

195357

studied the position of the double bond and the dimethyl amino group of

Conessine. Ganguly et al., 195358

isolated 0.01% of ß2sterol (C29H50O) similar to the ß

sitosterol from the unsaponified fraction of the kurchi bark. Alyn et al., 195759

reported the synthesis of benzo (C) phenanthrene-alkaloid of kurchi. Ram et al.,

196260

studied the action of nitrous acid on Holarrhimine.Bhattacharya et al., 196261

studied the synthesis of Conessimine from Conessine. Rudolf et al., 196362

isolated

Holadysone-11a-20-dihydroxy-18-20-epoxypregna-1-4-dien-3-one, and glycosides,

stigmastadienol, stigmastenol and ergostenol from kurchi bark. Mansa and

Bhattacharya et al., 196463

studied the structural corelation of Holarrhimine and



paravallarines. Godse et al., 196364

studied the effect of neighbouring groups in

derivatives of Holarrhimine. Victor et al., 195365

detected the presence of L-

Quebrachitol in kurchi. Labler et al., 196666

isolated some secondary formed weak

bases like (20-R)-3a-(dimethylamino)-18,20-oxidopregn 5-ene-20-one and carbonyl-

N, -N-bis (3ß –dimethyl amine –N-dimethyl Conan_5_ene), from kurchi. Powell et

al., 196967

isolated 9-D-hydroxy-cis-12-octa decenoic acid from the seed oil of kurchi.

The known occurance of this acid limited to the genus strophanthus. Bhattacharjee

and kapoor et al., 196968-69

and reported presence of terpenes and alkaloids and the

absence of sterol, saponins, tannins, and flavanoids in the stem bark of kurchi.

Gouteral et al., 197070

review the new type of gluco alkaloids, the

aminoglucosteroids, isolated from the Asiatic species of the family Apocynaceae.

Daniel et al., 197871

detected 2.3% of tannins in the leaves of kurchi. Thanki and

Thaker et al., 195072

isolated about about 15 amino acid from the seeds of kurchi, the

dominant amongst them being aspartic acid and arginine. The amino acid content of

the protein hydrolysate of the seed was found to be comparable with that of groundnut

seeds. Narayan and Naik et al., 198173

isolated triterpine, 5,20(29)-Lupaddien-3 ß –

ol, a first natural product known with a double bond in ring B of the lupane skeleton

from the bark of kurchi. They also isolated from the bark Singh et al., 198374

studied

the leaf protein of kurchi. The wealth of India (1959) mentioned the composition of

certain constituents of kurchi like gum, seed oil, latex, etc, and described their

standards.

Schroff and dhir et al., 193975

developed an assay for kurchi and kurchi bismuth

iodide. Karkun and goha et al., 194376

proposed a method of analysis of kurchi

alkaloids which later on, was included in IPL. Basu and mithal et al., 194777

investigated the thermolabile and alkali unstable conditions of kurchi alkaloids and

indicated the defects of IPL method which mentioned prolonged heating and

association of alkaloids with alkali. Basu and mithal et al., 194878-79

better method

for the assay of kurchi alkaloids using ethanol: chloroform (1:3) containing 2%

ammonia as solvent for extraction at 500 c. Rao et al., 1948

80 and Basu and

Bhattacharya et al., 194981

proposed volumetric methods for estimation of kurchi

alkaloids. All these methods are comparatively studied by (Ghosh et al., 1949)82

.



Piette et al., 194983

validated the method of (Mascre and Loiseau et al., 1941)84

and

used it for estimation of Conessine in pure solution. Ommen et al., 195185

reported

43% of thermostable and 57% thermolabile types of alkaloids in kurchi. (aishankar

and Basu et al., 196186

and Labler and Cerny et al., 196387

separated kurchi

alkaloids by paper chromatography and thin layer chromatography respectively.

Vishin and gupta et al., 196788

estimated the alkaloids of kurchi bark by non-aqueous

titrimetry. Jayswal and Basu et al., 196789

estimated Conessine

spectrophotometrically in kurchi and W.tomentosa bark. Khorana and Vasudevan et

al., 196790

devised a method for the estimation of Conessine in the formulation of

kurchi bark. Dwivedi and Sharma et al., 199091

developed a turbidimetric method for

the quantitative estimation of total alkaloids of kurchi bark in crude medicinal

preparation and in the body fluid of man and rat.

Gupta and Sen gupta et al., 194692

utilized diastase in the extraction of alkaloids and

obtained an increased % of alkaloids from 0.6 to1.18% in the chloroform extract of

kurchi bark. The degrading action of diastase was specified on cellular matter, and

made the cell wall more permeable to organic solvents. Thakkar et al., 197293

employed ultrasonic energy for the extraction of the alkaloids from kurchi bark.

Sharma and Bal et al., 195994

studied the effect o the extract of kurchi bark on plant

tissue. Daniel et al., 1978a95

studied the chemotaxonomy of Apocynaceae. Royal et

al., 198896

reported the presence storage fungi Aspergilus flavun in kurchi and studied

their mycotoxins (1990). Shyam et al., 198997

studied the copper accumulating ability

of kurchi.

Brown et al., 192298

reported very good antidiarrhoeal effect of the seeds extract of

Apocynaceae plants containing Conessine, in case of chronic amoebic dysentery.

Chopra et al., 192799

have reported number of pharmacological action of Conessine,

like its feeble toxicity on protozoal flaggellates such as Trichomonas hominis,

inhibition on the activity of digestive enzyme such as pepsin and trypsin, cardiac

irregularities in large doses and its toxic effect on Entamonas histolytica. The toxicity

of Conessine, Holarrhine, and Oxyconessine was exhibited in vivo on tubercle bacilli

(Meissner et al., 1930)100

. Chopra et al, 1933101

studied the pharmacological action

action Kurchicine and found it to be protoplasmic poison like emetine. The alkaloids



simulated the plain muscles of the intestine and uterus, dilated the vessels of the

splenic area, produced a fall blood pressure and has a direct depressant action on the

heart, in particular the auricular ventricular bundle. Bakhsh et al, 1936102

determined

the lethal doses of Conessine, Kurchicine and iso Conessine and studied the various

pharmacological actions of these alkaloids. Conessine raised (in small dose), and

Lowered (in large dose) the B.P., contracted the renal vessels, dilated the intestinal

vessel but did not have any effect on the coronary vessel of isolated rat heart.

Isoconessine was less toxic in comparison of Conessine and showed a more marked

stimulating effect on frogs voluntary and smooth muscle of intestine and uterus.

Siddiqui et al, 1936103

carried out a comparative pharmacological study of Conessine,

Isoconessine, and Neoconesine.

Alfered et al, 1944104

showed that Conessine, Conessidine, conkurchicine, kurchicine

and holarrhenine in high dilutions, kill paramecia, colpidia and daphnia, like emetine.

Jones et al, 1947 and Pitette et al., 1949105

compared the amoebicidal properties of

Conessine and emetine in vivo. Lavier et al., 1948106

reported the antiamoebic

spectacular activity of Conessine ad showed it to possess good results with negligible

side effect like trembling, nightmares or insomnia. Duriex et al., 1948107

reported

spectacular antidiarrhoeal activity of Conessine in cases of primary and secondary

infections where emetine failed to work. (ipette et al., 1950108

and Auffret et al.,

1950109

studied the accumulating property of Conessine in different organs of

experimental animals. Pluchon et al., 1950110

stated that sub-therapeutic doses of

Conessine are also prone to get fixed in various organs like spleen, lungs, liver,

kidney, brain, and could be detected letter.

Lambir, Bernard et al., 1953111

and Mukerji et al., 1953112

observed in vitro, the

inhibiting action of Conessine on the growth of Mycobacterium tuberculosis. The

Wealth of India, 195916

mentions good result of a glycerin suppository containing

Conessine hydro bromide in cases of trichomoniasis. Jaishankar et al., 1961113

studied the general pharmacological action of conkurchicine and reported it to be

devoid of any significant effect on the isolated rectus and abdominis of frog, ileum,

uterus, ileum, and CNS of the rat, but decrease the heart rate in case of higher doses.

Bhavsar et al., 1965114

studied the fresh juice of kurchi leaves for its bacteriostatic



activity against S. aureus and E.coli and found it. Basu and Jayswal et al., 1968115

tested Conessine in vitro, against the “c” strain of Entamoeba histolytica and reported

it to be a more potent amoebicidal agent in comparison to Conessine dihydrate,

conkurchicine, holarrhenine, Holarrhine and kurchicine. Dhar et al., 1968116

studied

the pharmacological activities of the alcoholic extract of stem, fruits, and stem bark

and reported the former two to possess antispasmodic activity on isolated guinea pig

ileum but did not antibacterial and antifungal activity and gross behavioral effecting

mice. Singh and Singh et al., 1972117

found that the bark extract increased the lesion

number against potato virus x (PVX). Raj et al., 1974118

showed that the stem bark

extract did not have any effect on human Ascaris lumbricoides in vitro. Nandi and

Mazumdar et al., 19798 reported the maximum antispasmodic activity of the

homeopathic tincture o kurchi bark prepared by using 70% alcohol. Deshmukh and

Jain et a., 1981119

mentioned that the seeds oil of kurchi showed a homeopathic

keratinophillic fungus, like Chrysopsorium indicum, C.pannicola, Malbroanchea

aurentica, Keratinomyces ajelloi and Microsporum gypseum

Clinically, polyherbal Ayurvedic formulation containing stem bark of kurchi as one

ingredient possessed good antidysentric, Singh and Chaturvedi et al., 1981, 1982a121

and dysenteric and diarrhoeal properties Javalgekar et al., 1982122

.

Chaturvedi and Singh et al., 1983123

conducted study on side effect of kurchi bark

powder in 11 indoor patient and observed that the drug can lead to subjective

symptoms as well as to hypo tension. Abrol and Chopra et al., 1965124

reported the

negligible inseticidal activity of alcoholic extract of the bark against houseflies and

mosquitoes. Suryakala et al., 1983125

has studied the juvenomimetic activity of the

extract of the stem of Dysdercus similes, Spodoptera liture, Musca domestica and

Anopheles stephensi and showed it to possess a gonadotropic effect on the females,

extract in A. stephensi. Thappa et al., 1989126

have observed a wide range of

insecticidal property of Conessine 0.5 to 10ppm dose against Aedes aegypti,

Dysdercus koenigit, Spodoptera litura and Pcris brassicae species. The seed and bark

of kurchi contain almost the same chemical constituents, but seed alone have been

proved to possess hypoglycemic activity and not the bark. Dhar et al., 1968116

and

Khanna et al., 1981131

. The authors personally communicated with the tribal people



of Gujarat residing around surat and junagadh district and found them to be using the

decoction of the seeds of kurchi in cases of diabetes mellitus, but certain Ayurvedic

antidiabetic formulations like Nyagrodhadi churna (Swami et al., 1950)127

, Asanadi

ghanvati (Pandya et al., 1991)128

, phaki (Ainapure et al., 1985)129

, etc. incorporating

the seeds of kurchi are known. Hence, the authors underlook screening of seeds of

kurchi for their hypoglycemi9c and antidiabetic activity. The aqueous and alcoholic

extract s (95% Ethanolic) of the drug at a dose of 250mg/kg body weight P.O were

tested for their effect on the blood sugar level on albino rats buy normal fasted model

and glucose loaded model. Both the extract exhibited significant hypoglycemic effect

in both the models. The extracts were then tested on streptozotocin induced

hyperglycemic rats at the same dose level and was found to produce significant

hypoglycemic activity (Gopal and Chauhan et al., 1993)130

. The seed and bark

contains almost the same chemical constituents, but seeds have alone proved to

possess hypoglycemic activity and not the bark (Dhar et al., 1968)116

. These

suggested the possibility of some constituents other than alkaloids responsible for the

hypoglycemic activity of the seeds of kurchi. Proteins like insulin and polypeptide P

Khanna et al., 1981131

have been proved to possess good hypoglycemic activities.

Hence the hypoglycemic property of primary metabolites was thought worth to

investigate. The protein fraction of kurchi seeds at a dose of 100mg/kg body weight

i.p in normal fasted model and glucose loaded model of albino rats did not reveal

significant hypoglycemic activity (Trivedi et al., 1991)132

. The hypoglycemic

activities of the other fraction of the seeds are under investigation.

Heble et al., 1971133

isolated 24-methylene cholesterol from callus tissue raised from

germinated seedling of kurchi and established callus tissues and various cell lines.

Heble et al., 1973134

studied the effect of various phytochormones such as IAA, NAA

and cytokinins on the growth and production of secondary constituents of callus

tissue, and noticed significant accumulation of the metabolites of cholesterol like 24-

methylene and 28-isofurastane and the inhibition of Conessine synthesis in callus

tissue of kurchi. Based on these facts they have suggested that a modification of

steroid metabolism under cultural condition is possible (Heble et al., 1974)135

. When

cholesterol-4-c14

was administered to 10 days old callus, radioactive 24-methylene



cholesterol, 28-isofucosterol, sitosterol, stigma sterol, and Conessine, were produced,

there by indicating the conversion of cholesterol in sitosterol mediating through 24-

methylene cholesterol and 28-isofucosterol in this system (Heble et al., 1976a)136

.

Callus cultures derived from the hypocotyls of germinated seedlings of kurchi showed

an inherent lack of organ forming ability when grown under the influence of a wide

range of exogenous growth factor. A number of sterol were isolated from the callus, of

which the predominant once were identified as cholesterol, 24-methylene cholesterol,

28-isofucosterol, sitosterol and stigmasterol (Heble et al., 1976)137

. Several sterols and

steroidal alkaloids were dected by them in the suspension culture of kurchi m (Heble

et al., 1977)138

.

Panda et al., 1991139

established callus and suspension culture of kurchi for the

production steroidal alkaloids especially Conessine. The doubling time and specific

growth the rate of cells in suspension culture were computed to be 47.5hr. and 0.35 hr

per day respectively. A maximum of 300 mg alkaloid per 100 g dry cell wt in 40 days

and 130 mg per 100 g dry cell wt in 8 days were obtained in the callus and suspension

culture respectively. About 90% of the total alkaloids produced in on the growth and

alkaloid production. A modified murashige and skoog (MS) medium that contains

60mm total N with a nh+4 to NO-3 ratio of 5:1, 0.25 mm phosphate and 4g/L sucrose

was developed for increasing the yield of Conessine. The growth regulators 2,4-D and

kinetic were found to affect the alkaloid synthesis. Using an optimal level of inoculam

(3g/L). The modified medium resulted in alkaloid synthesis of 0.66g/100g dry cell wt:

which represented a 4.25 fold increase over that obtained in standard M.S medium

(Panda et al., 1992)140

. A precursor feeding strategy for increasing the yield of

Conessine in cell suspension culture was also established by them. A total of 50mg/L

of added cholesterol was converted to 43mg/L of alkaloids 90% of which was

Conessine. By applying the precursor in 8 days. In this way the alkaloid content of the

cell were increased 76 fold compared to that obtained in the standard MS medium.

The step leading to biotransformation of cholesterol to alkaloids were unaffected by

phosphate. The shake flask data were successfully transferred to a bench scale 6L

stirred tank bioreactor in which the biosynthesis rate of alkaloid production was



110mg/100g dry c3ells 160 fold higher than that of whole plant (Panda et al.,

1992a)141

.

Rajashekar et al., 1973142

have established a method for isolating the protoplast from

the culture plant cells of kurchi. Various factors affecting the release of protoplast

from the cell like effect of pectinase, age of cell, effect of organic and inorganic

sodium salt etc. were studied, further they have reported morphological observations

of the isolated protoplasts, also. Dohnal Barbara et al., 1990143

studied one and six

year old callus tissue of kurchi and reported five alkaloids, two of them being as

Conessine and conimine. The alkaloid extract was found to inhibit the growth of

Shigella sonnei, Sh. Flexneri and Salmonella enteritidus strains but not of S, typhi and

S. paratyphi. Kulkarni et al., 1992144

have worked on the in vitro, propagation of

kurchi. They found IAA (2mg/L) to be the most favorable, for including the callus in

root and stem and 2,4-D (0.5 mg/l) in leaf explants. The explants taken from the total

segments of stem regenerated to shoots on MS medium supplemented with IAA

(1mg/L) and on transferring it to medium containing 3mg/L of IAA, the shoots

developed roots leading of complete planets.



2.3 Ativish

The unripe fruit is acrid, astringent, carminative, and tonic. The ripe fruit is

demulcent, nutritive, laxative, cooling, fattening, and useful in biliousness, the seeds

are diuretic. The leaves are used in skin diseases. Ash of the leaves is useful in healing

ulcer. The root if said to be useful in skin disease, asthama, and diarrhea. Juice of the

plant is applied to glandular swelling and abcesses to promote suppuration. It is also

used for snake bite (Nandakari et al., 1954; Chopra et al., 1956, 1958; Wealth of

India 1985)7,15,16

.

The tree find use in toothache and caries, stomach complainnts, sores, carbuncle on

the back, sterility in woman and post natal complains (Jain and Tarafder et al.,

1970)145

the stem bark is used for application on eczema (Hemadri and Rao et al.,

1989)146

it finds use in epilepsy (Hembrom et al., 1983)147

headache (Das and Misra

et al., 1988)148

id in grandular swelling (Nautiyal and Nautiyal et al., 1983)149

the

bark is used as galactagogue (Sikarwar et al., 1992)150

the unripe fruit is acrid,

astringent, carminative, and tonic (Banerjee and Banerjee et al., 1986)151

. The ripe

fruit is demulcent, nutritive, laxative, cooling, fattening, and useful in biliousness, the

seeds are diuretic (Ahluwalia et al., 1969 and Jha et al., 19970)152-153

and as an

aphrodiasiac (Ahmad and Chaghtai et al., 1982)154

. The leaves are used in skin

diseases, as an antidote to snake bite and scorpion sting (Ahuwalia et al., 1968;

Nautiyal and Nautiyal et al., 1983)152,149

; (John et al., 1984; Reddy et al., 1988;

Balaji Rao et al., 1995; Ahmed et al., 1996)155-158

and as galactagogue (Sharma and

Sinha et al., 1980)159

. The root is used as laxative, in Diarrhoea and in skin diseases

(Ahluwalia et al., 1968; Nautiyal and Nautiyal et al., 1983; Chandra and Pandey

et al., 1985; Ahmad et al., 1996)152, 149, 160, 158

.

Table 2.2 Chemical constituents of Aconitum heterophyllum.

Chemical

constituents

Type of

compound

Part used References

Morin (γ, 5, 7, β’, 4’- pentahydroxyflavone

Flavon Heartwood (Perkin and Cope et al., 1895)

161

Dihydromorin Flavone (Chakravarty and

Seshadri et al., 1962)162

Cynomaclurin (5, 7,

β’, 4’ –tetrahydroxy-

3-ketoflavan

Flavone (Perkin and Cope et al.,

1895; Chakravarty and

Seshadri et al., 1962,



1963, 1964; Nair and

Venkatraman et al.,

1963; Nair et al., 1966)

163-166

(±) Cynomaclurin

Trimethyl ether

Flavone (Bhattia et al., 1966;

Nair and Venkatraman

et al., 1956; Dave et al., 1961, 1962)

167-169

Cycloartocarpin Flavone (Nair et al., 1964)170

Isoartocarpin Flavone (Dave et al., 1962)171

Artocarpetin (5, β’, 4’, - trihydroxy-7-

methoxyflavone)

Flavone (Dave et al., 1960,

1962)172

Artocarpesin (6-

prenyl-5, 7, β’, 4’ - tetrahydroxyflavone)

Flavone Young

Heartwood (Radhakrishnan et al.,

1965; Radhakrishnan

and Rama Rao et al.,

1966)173-174

Norartocarpetin (5, 7,

β’, 4’ - tetrahydroxyflavone)

Flavone (Radhakrishnan et al., 1965)

175

Ycloartocarpesin Flavone Heartwood (Parthasarathy et al., 1969)

176

Oxydihydrocarpesin Flavone Heartwood (Parthasarathy et al., 1969)

177

Cycloheterophylline Flavone Bark (Rama Rao et al.,

1971)174

Heterophylline Flavone Bark (Rama Rao et al., 1971)

175

Isocycloheterophylline Flavone Red power

under the

bark

(Rama Rao et al.,

1973)176

Artocarpanone

Artocarpetin (5, β’, 4’, - trihydroxy-7-

methoxyflavone)

Flavanone Red power

under the

bark

Red power under the

bark

Artoflavanone (5-

Hydroxy- 6-C—prenyl-7, γ’, 4’, 5’- tetramethoxy

flavanone

Flavanone Red power

under the

bark

Red power under the

bark

Kaempferol Flavonoid Fruits (Ganju and Puri et al., 1959)

162

Erioddictyol Flavonoid Fruits (Ganju and Puri et al., 1959)

162

Cycloartenone (earlier

reported as

Triterpenoid Latex,

Fruits, root, (Nath et al., 1935, 1937a,

b; Nath and Mukherjee



artostenone) leaves et al., 1939; Banerjee

and Bhattachariya et al.,

1945; Nath and

Chakraborty et al.,

1945; Nath et al., 1946;

Balakrishna and

Seshadri et al., 1947a,b,

1948; Mahato et al.,

1967b; Dayal sand

Seshadri et al., 1974;

Pant and Chaturvedi et

al., 1989; Barik et al.,

1994)177-189

Cycloartenol Triterpenoid Leaves and

Latex (Mahato et al., 1967b;

Barik et al., 1994)167, 189

Betulinic acid Triterpenoid Root (Dayal and Seshadri et

al., 1974)187

Ursolic acid Triterpenoid Root (Dayal and Seshadri et al., 1974)

187

9, 19-Cyclolanost-3-

one-24, 25-diol (24R)

Tetra cyclic

Triterpenoid

Dried Latex (Barik et al., 1994)189


one-24, 25-diol (24S)

Tetra cyclic

Triterpenoid


(24R)- and (24S)-9,

19-Cyclolanost-25-

ene-γ , β4-diol

Tetra cyclic

Triterpenoid



ene-3, 25-diol

Tetra cyclic

Triterpenoid


- sitosterol Sterol Leaves, root (Mahato et al., 1967b;

Dayal and Seshadri et al., 1974)

167-187

-D- galactose Monosacchariude Seeds (Suresh kumar et al.,

1982)191

Aurantiamide acetate Dipeptide Seeds (Chakraborty and

Mandal et al., 1981)192

4-Hydroxyundecyl

dpcpsenoate

Fatty acid ester Latex (Pant and Chaaturvedi

et al., 1989)193

The rind of the ripe fruit and the edible portion of the raw fruit mostly contained

fibrous materials containing calcium and pectin (Bhatia et al., 1955)194

. The fruit

were found to be devoid of 5-hydroxytriptamine (Sinha et al., 1961)195

. The fruit also

contained niacin (Sengupta et al., 1958)196

. The essential amino acids found in the

fruits are argenine, cystine, histidine, leucine, isoleucine, lysine, methionine,



phenylalanine, threonine, tryptophan and valine (Sengupta et al., 1958)196

. The

essential amino acids found in the phosphate and vitamin C contents of uncooked

vegetable were reported (Nanda et al., 1972)197

. Besides oxalic acid, calcium and

phosphorous contents were also determined (Singh et al., 1973)198

. The tenderfruits

contained sodium and potassium (Gopalan et al., 1971)199

. The fruit is widely

consumed as it is rich in -carotene (Chandrasekhar et al., 1999)200

.

A new marker haemagglutinating lectinn was obtained from the fruit (Chatterjee et

al., 1979)201

. The seeds revealed a powerful trypsin inhibitor, which could be

extracted with phosphate buffer or dilute hydrochloric acid. The activity of the extract

was destroyed completely by autoclaving it for about 30min and by boiling in the

water or salt solution or by backing (Siddappa et al., 1957)202

. The seeds are mostly

starchy and contain protein, calcium, and thiamine (Bhatia et al., 1955)194

. The seeds

also contained magnesium, sodium, potassium, copper, sulphur and chloride. The

oxalic acid and phytin contents in the seeds were determined. The essential amino

acids in the seeds were cystine, leucine, isoleucine, lysine, phenylalanine, methionine,

threonine, tryptophen anvaline (gopalan et al., 1971)199

. The seeds kernels contained

29.5 percent of starch on dry basis after purification. Enzymatic hydrolysis of the

starch indicated maltose as the end product, and it may serve as a source for the

industrial production of maltose (Kavith et al., 1992)203

. A unique α-galactose-

specific lectinn jacalin was also isolated from the seeds. It was a tetrameric

glycoprotein and had two saccharide binding sites (Suresh kumar et al., 1982 and

Basu et al., 1986)191, 204

.

Quantitative analysis of the leaves from Orissa yielded crude protein, crude fibre,

nitrogen free extract, total ash, calcium, phosphorous and tannins (Das et al., 1991)205

.

The leaves from west Bengal gave protein, hemicellulose, cellulose and permanganate

lignin. The total ashes, silica, dry matter, and neutral as well as acid detergent fibre

contents were also determined (Chakraborti et al., 1988)206

. The leaves being fairly

rich in crude protein and low in crude fibre, the digestibility coefficients of various

nutrients were comparatively low as is the case with many of the tree leaves. The high

tannin content of jack leaf, however, is a factor to be reckoned with in the feeding of

goats with liberal quantities of these leaves as fodder (Devasia et al., 1976)207

. The



latex from the system was found to contain four proteins (Pant and Srivastava et al.,

1965)208

.

Olive oil solution of artosterone, the new hydroxyl ketone prepared from artostenone,

when administered for 21d in sexually immature male rats increased (19%) the weight

of the prostate and seminal vesicles compared to that of the normal litter maters as

controls with a small dose of 0.05mg (50 ) per day. There was a decrease in the size

of testis, an accelerating in the rate of involution of thymus and a stimulating effect on

the kidneys. Histological examination revealed the opening of the central lumen of the

vas deferens. In the testes, the spermatogenic cells in the seminiferous tubules were

less in number and developed faster. Artosterone was suggested to be highly

androgenic in character (Nath and Sengupta et al., 1939)209

. Bioassay of leaves used

as animal feed was shown to have an estrogenic activity in mice . the activity was

found in the F1 fraction (Methanol soluble free estrogen) (Ray and Pal et al.,

1967)210

.

The seed extract agglutinated blood cells of various animals (Sathe et al.,

1967,1970)211, 212

. A new marker lectin obtained from the fruit, was studied with

respect to its haemagglutination pattern with various normal and enzyme treated red

cells and with special regard to their precipitin reaction with different

glycosubstances. The lectins reacted with special regard to their chain of Thomensen-

friedenreich (TF) type (3-0-ß-D-galactisamine) in serum and other glycoproteins, in a

similar but not identical way as the anti-TF peanut lectin (Chatterjee et al., 1979)201

.

The haemagglutinating matetrial from jack fruit was composed of two isolectins of

molecular masses 11500 and 15000. the lectins agglutinatred native red blood cells of

human A,B,) groups and sheep, rabbit and mouse erythrocytes. The lectins were

composed os a single polypeptide chains and they contained nocovalently linked

sugar. The lwer molecular mass material was present in considerably greater quality

than the higher properties of this lectin were studied (Vijayakumar et al., 1987)213

.

The presence of lectin was also shown by (Arora et al., 1987)214

in the plant.

A lot of work has been done at the regional cancer centre, thrivanathapuram, for new

tissue specific plant lectins and their potential in the histochemical and cytochemical

aspects of oncology, the jack fruit lectin (JFL), an N-acetyl-D- galactosamia specific



non glycosylated protein, isolated from the seeds was used as histochemical marker

for ethmoid carcinoma in bovines. The lectin was conjugated with horse radish

peroxidase (HRP). The binding to neoplastic tissue was compared to that of normal

controls. The neoplastic cells showed varying degree of binding of the JFL in contrast

to normal control which generally adopted uniform binding. The technique ciould be

useful in evaluating tumours in animals (Manu Mohan et al., 1998)215

.

The HRP conjugateds JFL was reported to be of diagnostic importance in carcinoma

cervix and in premalignant and makllignant lesions of oral cavity. The binding of the

JFL could be inhibited completely by N-acetyl-D-galactosamine. Histochemical

application of this electin in diagnostic and prognostic pathology have revealed that

the conjugated lectin was able to identify malignant tissues even before the clinical

signs are manifested (Vijayan et al., 1982,1987; Vijayakumar et al., 1987)216-217, 213

.

The binding pattern of this lectin has been studied in detail in beginning and malignant

lesions of breast and thyroid, carcinoma of the uterine cervix and in the exfoliative

cytology of bronchopulmonary neoplasia and cervical in patient (Remani et al., 1989,

1990, 1994; Pillai et al., 1992, 1994; Vijaykumar et al., 1992)218-223

. The

histochemical and cytochemical application of JFL in oncology was reviewed by

(Haseenabeevi et al., 1991)224

.

The mutagenic, co mutagenic and antimutagenic effects of selected food in items

including jack fruits where tested in Salmonella microsome assay using two mutant

strains TA98 and Ta100. thwe fruit were found to be both non mutagenic as well as

antimutagenic (Saroja at el., 1991)225

The expression of T-antigen in colon cancer tissue was detected by the T-alpha

specific plant lectin (ALL) from jack fruit. The lectin could localize the T-antigen in

6 of the 13 human colorectal adenocarcinoma tissues (46%) section tested. The lectin

and cost was found to be useful diagnostic value because of the ease of preparation

and cost effective ness (Sriram et al., 1999)226

.

The latex from the stem and young twins of jack tree exhibited bacteriolytic activity.

The protein content in the latex was found to be 48.94mg/ml while its activity was

325units/ml.in astudy on relationship between the lytic activity and the development

status of the plant part from which the latex was drawn, the lytic activity of the latex



from the young branch was nearly four times as high as that of the latex adrawn from

the main stem although their protein contents were same (Shukla and Krishnamurti

et al., 1961)227

.

The effect of the dietary fibre from the tender jack fruit has been studied on intestinal

mucosal and ß-glucuronidase activity in hexachlorocyclohexane (HCH) treated rats.

The feeding of the neutral detergent dietery fibre along with HCH revealed decrease

in ß-glucuronidase activity in the contents of cecum and colon and in the mucosa of

small intestine and colon as compared to rat fed with fibre free diet. The neutral

detergentfibre contained hemcellulose, lignin, cellulose, cutin and silica. The dietary

fibre decreased the biological activity of intestinal micr5oflora thereby decreasing the

absorption and reabsorption of HCH, the binding of HCH might be one of the reason

that result in the excretion of considerable quantities of HCH from the body (Serji

and Devi et al., 1993)228

.

The presence of an acetylcholine-like substance in the seeds of jack fruit was

demonstrated along with another active substance which had positive inotropic and

chronotropic effects on frog’s heart (Lal and Sreepathi et al., 1964)229

.

The seeds exhibited equal antitryptic and antichymotryptic activities against the

enzymes trypsin and chymotrypsin. However, these had no activity against subtilisin

enzyme. The inhibitory activities were generally more thermolabile under acidic

conditions. The activity was lost when the seeds were boiled in water or salt solution

or by baking (Sumathi and Pattabiraman et al., 1976)230

.

The 50 per cent ethanolic extract of the plant (excluding root) in a preliminary

biological study showed some CVS activity in dog/cat, while it was found devoid of

antibacterial, antifungal, antiprotozoal, antiviral, hypoglycaemic and anticancer

activities and effects on isolated guinea pig ileum, respiration, preganglionically

stimulated nictitating membrane and CNS in experimental animals. The extract of the

fruits showed antiprotozoal activity against Entamoeba histolytica strain STA. The

extract was devoid of antibacterial, antifungal, antiviral and diuretic activities and

effects on isolated guinea pig ileum, rat uterus, respiration, preganglionically

stimulated nictitating membrane, CVS and CNS in experimental animals. The LD5o



of the two extracts was found to be > 1000 and 825 mg/kg i.p., respectively in mice

(Bhakuni et al., 1969, 1988)231, 232

.

The 80 per cent Ethanolic extract of the leaves did not reveal antibacterial activity

against Escherichia coli, Pseudomonas aeruginosa. Bacillus subtilis and

Staphylococcus aureus strains using the agar dilution method (Valsaraj et al.,

1997)233

. The latex did not reveal marked fungitoxicity against Aspergillus aculeatus,

A. niger, Alternaria altemata, A. solani, Myrothecium roridum, Fusarium solani,

Penicillium expansum and Ulocladium chartarum (Sharma et al., 1994)234

.

The shoots revealed nematicidal activity against various nematodes viz; Rotylenchulus

reniformis, Tyienchorhynchus brassicae, Tyienchus filiformis and Meloidogyne

incognita. The aqueous extract of the leaves showed in vitro activity against

Meloidogyne incognita (Siddiqui et al., 1987, 1992; Sharma and Trivedi et al.,

1995)235-237

.



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as a histocheinical marker for ethmoid carcinoma in bovines. Int J Anim Sci 13;

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216. Vijayan K.K, Vijayakumar T, Sasidharan V.K. and Vasudevan D.M. Tissue

specificity of certain plant haemagglutins (lectins). Proc Recent Trend hnniuno

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217. Vijayan K.K, Remani P, Haseenabeevi V.M, Ankathil R, Vijayakumar T,

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218. Remani P, Augustine J, Vijayan K.K, Ankathil R, Vasudevan D.M, Krishna Nair

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219. Remani P, Joy A, Vijayan K.K, Ravindran A, Haseenabeevi V.M, Vasudevan

D.M. and Vijayakumar T. Jack fruit lectin binding pattern in carcinoma of the

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220. Remani P, Pillai K.R, Haseenabeevi V.M, Ankathil R, Bhattathiri V.N, Nair M.K.

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neoplasia. In Vivo 6; 1992; 107-112.

222. Pillai R.K, Remani P, Kannan S, Mathew A, Sujathan K, Vijayakumar T. and Nair

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223. Vijayakumar T, Augustine J, Mathew L, Aleykutty M.A, Balaraman Nair M,

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Chapter-3 Aim of the work

S.K.P.C.P.E.R (M.Pharm Dissertation) 50 Arun M Prajapati

Chapter 3

Aim of the work

The Ayurvedic and other herbal formulations are very popular in India. The growth of the

herbal medicines and food supplements has been very impressive world wide and quite

phenomenal. The share of Indian Herbal Medicinal Plants and Ayurvedic formulations in

the world market is very unimpressive. This is due to a number of lapes like; the active

compounds responsible for the proposed therapeutic activity are not properly identified

and quantified, there is no uniformity in the process of manufacture, no standard

operating procedure are available for production, analysis and validation of these, and

most important one is the formulation part, which is almost untouched. Furthermore,

there is also a lack of analytical procedures for the assessment of in vitro drug release of

active constituents from these drugs and formulations. In addition to these, the regulatory

agencies in India and abroad recently introduced the guidelines of GMP and WHO to be

incorporated and followed compulsorily for the manufacturing and trade of these drugs

and formulations.

Looking to these context, it is necessary to evaluate and standardize the existing

Ayurvedic formulations for their pharmacognostic and other pharmactotechnical aspects.

Hence, in the present investigation, a well known anti-dysenteric Ayurvedic formulation

“Kutaj Ghanvati” is selected for the study. It is aimed to evaluate these formulations in

following parameters.

Pharmacognostic and physico-chemical evaluation of raw materials

Analytical method development for the assessment of active ingredient for the

raw materials

Preparation of Kutaj Ghanvati

Pharmacognostic and physico-chemical evaluation of Kutaj Ghanvati

Analytical method development for the simultaneous estimation of active

constituents of the Kutaj Ghanvati

Pharmacotechnical evaluation of Kutaj Ghanvati

Chapter-3 Aim of the work

S.K.P.C.P.E.R (M.Pharm Dissertation) 51 Arun M Prajapati

In pharmacognostical evaluation, the macroscopical and microscopical characters of the

raw materials and formulation are studied. In physico-chemical evaluation, the

quantitative values such as foreign matter, extractives and ash values are determined by

the standard guidelines. Quantitative estimation of total alkaloids of Kutaj Ghanvati and

its ingredient drugs are carried out by the pharmacopoeial methods. Specific chemical

marker compounds of the ingredient drugs and formulation of Kutaj Ghanvati are

assessed by the proposed method for specific and simultaneous methods by HPTLC. In

Pharmacotechnical evaluation the study is extended to determine the tablet parameters

such as friability, crushing strength, disintegration time and in vitro dissolution study of

the laboratory sample and marketed formulations of Kutaj Ghanvati.

Chapter-4 Evaluation of raw materials

S.K.P.C.P.E.R (M Pharm Dissertation) Arun M Prajapati 52

Chapter 4

Evaluation of raw materials

4.1 Introduction

Efficacy and safety of the herbal drugs and formulations including ayurvedic and other

traditional formulations are always under a big question mark. These are mainly

concerned with the quality of raw material and methodology adopted during the

procurement, handling and processing them. The prime importance is given to the

standardization of the raw material which are used in such formulations to ascertain the

efficacy and safety. Many guidelines are published especially by WHO, Indian Herbal

Pharmacopoeia, Ayurvedic formulary and many others for the standardization and

evaluation of these materials.

Hence, in the present investigation the attempt was made to evaluate the raw materials

under the study for the pharmacognostic and physicochemical parameters according to

these guidelines. Also, the detailed methodology is also described to find out the amount

of active constituents present in these raw materials.



4.2 Experimental

4.2.1 Pharmacognostic and physicochemical evaluation of Kurchi and Ativish1

4.2.1.1 Materials

Two different samples of Kurchi bark and Ativish root were procured from the two

different herbal drug suppliers. Both the samples were powdered and passed through 60

mesh sieve. Powdered drug sample of kurchi bark and Ativish root were also procured

from the well known ayurvedic drug supplier, L. V. Gandhi & Sons, Ahmedabad.

Standardization of these entire powdered samples was carried out for the usual

pharmacognostic and physicochemical parameters as described in the methods.

4.2.1.2 Foreign matter1

100 g sample of the powdered plant material was spread in thin layer and sorted for

foreign matter in to groups by visual inspection, using a magnifying lens (10×). The

remainder of the sample was shifted through a sieve No 250. Dust was regarded as

mineral admixture. The portion of this sorted foreign matter was calculated as the content

of each group in grams per 100 gm of air-dried sample.

4.2.1.3 Macroscopic and Microscopic examination1

Since, the material is powdered sample macroscopical study for both the drugs was

skipped off. Powder characteristics of the drug were studied under the microscope. The

stained and unstained slide was prepared and the characters were examined and

photographed using CCD camera.

Method

1) About 1-2 gm powders was taken and dissolved in methanol, shaken for few minutes

and then it was filtered. The filtrate which contain extractable matter and chemical,

while the impurities and powder material were remained on the filter paper. Residue

was boiled with chloral hydrate foe few minutes. After boiling the power it was stained

with the HCL and phloroglucinol and at last washed with water. The powder was

mounted on the slide with lactophenol and covered with the cover slip. The slide was

examined under the microscope.



2) Unstained slide was also prepared as above and examined.

3) The iodine stained slide was also prepared and examined under the microscope.

4.2.1.4 Determination of Ash Value1

Total ash

4 g of the ground air-dried material was taken and accurately weighed, in a previously

changed and tared crucible (silica). The material was placed in an even layer and ignited

by gradually increasing the heat to 500-600 0C until it appeared completely white,

indicating the absence of carbon. The material called ash was cooled in a desiccator and

weighed. Again the residue was moistened with about 2 ml of water and dried on a water-

bath, then on a hot plate and ignited to constant weight. The residue was cooled in a

desiccator for 30 minutes and then weighed without delay. The content of total ash was

determined with respect to air-dried plant material.

Acid-insoluble ash

To the crucible containing the total ash, 25 ml of HCL (~70g/l) was added, covered with

a watch glass and boiled gently for 5 minutes. The watch glass was rinsed with 5ml of hot

water and washing was added to the crucible. Insoluble matter was collected on an ash

less filter paper and washing of this filter paper was carried out with hot water until the

filtrate was remaining neutral. The filter paper containing the insoluble matter was

transferred to the original crucible, which is then dried on a hot plate and ignited to

constant weight. Allowed the residue to cool in a suitable desiccator for 30 minutes, and

then weighed without delay. The content of acid-insoluble ash was calculated with

respect to the weight of air dried powdered plant material.

Water-soluble ash

To the crucible containing total ash, 25 ml of water was added and boiled for 5 minutes.

Insoluble matter was collected on an ash less filter paper. The residue was washed with

hot water and ignited in a crucible for 15 minutes at a temperature not exceeding 450 0C.

The weight of the residue was substracted from the weight of total ash. The content of



water soluble ash was determined with respect to the weight of the air dried powdered

plant material.

4.2.1.5 Determination of extractives1

Method 1: Hot extraction with water

Accurately weighed 4.0 gm of coarsely powdered air-dried material was taken in glass-

stoppered conical flask. One hundred ml of distilled water was added and weigh to obtain

the total weight including the flask. The flask was shaken well and allowed to stand for 1

hour. The content was refluxed for 1 hour ant then cooled and weighed. The weight was

readjusted to the original total weight with distilled water. The flask was shaken well and

filtered rapidly through a dry filter. Transferred 25 ml of the filtrate to a tared flat-

bottomed dish and evaporated to dryness on a water-bath and then dried at 105 0C for 6

hour. The extract was cooled in a desiccator for 30 mins, and then weighed without delay.

Percent water soluble extractive value was calculated with respect to the weight of the

air-dried material.

Method 2: Cold maceration with ethanol

Accurately weighed 4.0 gm of coarsely powdered air-dried material was taken in glass-

stoppered conical flask. The content was macerated with 100 ml of ethanol for 6 hour

with frequent shaking and then allowed to stand for 18 hours. The content was filtered

taking care not to lose any solvent. Transferred 25 ml of filtrate to a tared flat-bottomed

dish and evaporated to dryness on a water-bath and dried at 105 0C for 6 hour, cooled in

desiccators for 30 mins and weighed without delay. Percent ethanol soluble extractive

value was calculated with respect to the original weight of the air-dried material.

4.2.2 Determination of total alkaloids of kurchi 2

Total alkaloid was determined according to IP 1955.

Procedure

5 gm of kurchi powder was weighed and moistened with 10 ml solution of alcohol:

chloroform (1:3) containing 2% v/v of strong solution of ammonia for 15 minutes in glass

percolator. Macerated with more of solution of alcohol: chloroform (1:3) for an hour and



collected 25 ml of the percolate in a receiver containing 1 g of oxalic acid dissolved in 5

ml for the percolation, 10 ml of solution of alcohol: chloroform (1:3) containing 1% V/V

of sodium hydroxide, and macerated for 15 minutes. Continued the percolation by adding

further quantities of alcohol: chloroform (1:3) solution until the drug is exhausted. It was

mixed and percolated well and extracted well by shaking with 20 ml portion of 2N HCL.

The acid extract was combined and made alkaline with strong solution of ammonia

extracted with four 10 ml portion of solution of chloroform and added 1 ml of 2N NaOH

and extracted again with chloroform. Each chloroform extract was washed with same 10

ml portion of water continued in different separator. Combined the chloroform extracts

and added 20 ml of N/10 sulphuric acid and shaken well for 5 minutes. Chloroform was

washed with two 20 ml portion of N/20 Sulphuric acid. Then this portion was collected

and titrated using 0.1 M NaOH using phenol red as indicator, which show colour change

from red to light green. Total alkaloid was estimated using the factor given in the IP.

Factor Each ml of N/10 H2SO4is equivalent to 0.01657 go f total alkaloids of kurchi.

4.2.3 Estimation of total alkaloids of Ativish3

Total alkaloid was determined according to IP 1966.

Procedure

6 gm fine powder was weighed and accurately transferred to a 159 ml flask, added 60 gm

of solvent ether and 2.5 ml of diluted with ammonia solution and shaken vigorously at

frequent intervals during thirty minutes. Added 25 ml of water, shaken and allowed to

stand, decent, filtered through cotton wool.40 gm of ethereal solution was taken, which is

representing 4 gm of aconite powder in 150 ml conical flask and evaporated to dryness

on a water bath. 5 ml of ether was added and evaporated to dry ness on a water-bath and

repeated this process with further 5 ml of ether. 5 ml of alcohol was added and heated on

water-bath for five minutes. 30ml boiled and cooled water was added in it. 8 drop of

solution of methyl red and 1 drop of 0.1 %W/V solution of methylene blue in alcohol and

titrated with 0.1N HCL to pink-Violet colour. Total alkaloid was calculated by using

factor given in the IP.

Factor Each ml of 0.1N HCl is equivalent to 0.0645g of total alkaloids, Calculated as

aconitine.



4.2.4 Estimation of conessine in Kurchi by HPTLC4.

4.2.4.1 Material and chemicals

Conessine standard

Kurchi powder

Methanol

Diethyl ether

Chloroform

Toluene

Ethyl acetate

Diethyl amine

Ammonia

Dragon-droff’s reagent

Sodium nitrite 10% Aqueous solution

Distilled water

Conessine reference standard was kindly gifted from Cadila Pharmaceutical Ltd, Dholka.

4.1.4.2 Instrumentation

Analysis was performed on 10cm x 10cm plates cut from 20cm x 20cm aluminium-

backed silica gel 60 F254 plates. Samples were applied to the plates by means of a

Linomat-V automatic spotter with the aid of Hamilton 100 µl syringe. TLC plates were

developed in flat bottom twin trough chamber. Densitometry was performed with a TLC

scanner-3 with Win CATS 4 software resident in a Pentium IV computer.

4.2.4.3 Chromatographic condition

Stationary phase: Methanol prewashed 10cm x 10cm aluminium-backed silica gel

60 F254 plates (E.Merck)

Mobile phase: Toluene: Ethyl acetate: Diethyl amine (6.5:2.5:1)

Chamber saturation: 30 minutes

Band width: 6 mm

Distance between tracks: 11.4 mm

Rate of spotting 10 sec/µl



Distance run: 80mm

Spraying reagent: Dragondroff was sprayed after drying the plate and then

sprayed 10% solution of aqueous sodium nitrite, plate was dried in air and after

20 minutes plate was scanned.

Scanning Wave length: 520nm

Scanning speed: 5mm/sec

Slit dimension: 5.0 X 0.45mm

Temperature: 25 0C

4.2.4.4 Preparation of standard solutions

Conessine stock standard solution was prepared by weighing and diluting 10 mg of

standard conessine up to 100 ml with methanol. 1 ml of this solution was taken and diluted

up to 10 ml with methanol to bring the solution of 10 μg/ml.

4.2.4.5 Preparation of sample solutions

1 gm of powder of each sample of kurchi was refluxed with 20 ml of a mixture of diethyl

ether: chloroform (3:1) and 1 ml of 10% ammonia solution. Filtered, evaporated and

dissolved the residue in 40 ml methanol. 1 ml from this was taken and diluted up to 10 ml

with methanol.

4.2.4.6 Preparation of standard curve

Analysis was performed on 10 cm 10 cm precoated silica gel 60 F254 TLC plate (E.

Merck) of uniform thickness Plates were prewashed by development with methane then

dried in a air. For preparation of the standard curve apply 1 to 6 µl volumes of the diluted

TLC standard solution (10-60 ng) spotting was done by Linomat-V spotter. The plates

were dried in air and Conessine detected by spraying lightly and evenly (not to wetness)

with Dragondroff and then dried at room temperature in the air. After drying the plate

Sodium nitrite was sprayed on it and again dried in air for minimum of 20 minutes and

then Standard zones were scanned at 520 nm with Scanner-3 as mentioned the

chromatographic condition above.



4.2.4.7 Estimation of conessine

From the sample solution 2.5, 3.5 and 4.5µl was applied on the precoated silica gel plate

and process was repeated to develop and scan the plate as mentioned above. A calibration

equation relating to the standard concentration to scan areas was determined by the use of

a linear regression program on a personal computer, and the amount of conessine in the

samples was calculated from the calibration equation by using the average area of

triplicate sample aliquots. The results were recorded.

4.2.4.8 Validation of the HPTLC method5

The method was validated as per ICH guidelines for Linearity, Precision, Limit of

Detection, Limit of Quantitation, Accuracy and Specificity

Linearity

Linearity of the method was performed by analyzing standard solution of conessine by

the proposed method in concentration range 10 to 60 ng/spot.

Accuracy

Accuracy of the proposed method was determined by recovery study. Recovery study

was carried out by adding three different quantities of conessine (10, 15, and 20 ng/spot)

to preanalyzed solution of sample of Kurchi bark-2 containing 10 ng/spot. All the

procedure was repeated five times as discussed above. From the linear regression

percentage recovery of conessine was determined.

Precision

Precision was determined by repeatability, intra day and inter day reproducibility

experiment of the proposed method. Repeatability was evaluated for degree of

repeatability of spotting by preparing and analyzing the standard solution of the drug six

times. The intra day reproducibility was determined by analyzing freshly prepared

solution in triplicate at three different concentration whereas inter day reproducibility was

checked by analyzing the standard solutions at six different days under same operative

condition.



Limit of Detection and Limit of Quantification

Limit of detection and Quantification of conessine was calculated visually by error and

trial.

Specificity

Specificity of an analytical method is its ability to measure the analyte accurately and

spefically in the presence of component that may be expected to be present in the sample

matrix. 30 µg of test and standard conessine were spotted on the TLC plate, developed

and scanned as described above. The test chromatogram was compared with the standard.

4.2.5 Estimation of atisine in Ativish by HPTLC4

4.2.5.1 Materials and chemicals

Atisine standard

Ativish powder

Methanol

Chloroform

Toluene

Ethyl acetate

Diethyl amine

Dragondroff reagent


Distilled water

Atisine reference standard was kindly gifted from Cadila pharmaceutical LTD, Dholka.









4.2.5.3 Chromatographic conditions



Mobile phase: Toluene: Ethyl acetate: Diethyl amine (7:2:1)


Band width: 6 mm



Distance run: 80mm

Spraying reagent: Dragondroff was sprayed after drying the plate and then



Scanning Wave length for UV: 274 nm

Scanning Wave length for Visible: 520nm



Temperature: 25 0C


Atisine stock standard solution was prepared by weighing and diluting 10 mg of standard

Atisine up to 100ml with absolute methanol. 1 ml solution of it was taken and diluted up to

10 ml with methanol to bring 10 µg/ml concentration.


1.0 gm of powder of each sample of Ativish was extracted with 10 ml of methanol.

Filtered, evaporated and dissolved the residue in 40 ml methanol. 1 ml from this was

taken and diluted up to 10 ml with methanol.







TLC standard solution (10-60 ng) spotting was done by Linomat-V spotter. The plate was

dried in air and late was scanned at 274 nm. atisine show maximum absorption at 274

nm. atisine was also detected by spraying lightly and evenly (not to wetness) with

Dragondroff and dried at room temperature in the air. After drying the plate Sodium

nitrite was sprayed on it and again dried in air for minimum of 20 minutes and then

Standard zones were scanned at 520 nm with Scanner-3 as mentioned the

chromatographic condition above.

4.2.5.7 Estimation of atisine

From the each of the sample solutions 2.5, 3.5 and 4.5µl was applied on the precoated

silica gel plate and process was repeated to develop and scan the plate as mentioned

above. A calibration equation relating to the standard Concentration to scan areas was

determined by use of a linear regression program on a personal computer, and the amount

of Atisine in the sample was calculated from the calibration equation by using the

average area of triplicate sample aliquots. The results are recorded



Detection, Limit of Quantitation, Accuracy and Specificity.

Linearity

Linearity of the method was performed by analyzing standard solution of Atisine by the

proposed method in concentration range 10 to 60 ng/spot.

Accuracy

Accuracy of the proposed method was determined by recovery study. Recovery studies

were carried out by adding three different quantities of Atisine (10, 15, and 20 mg) to

preanalyzed solution of Sample (Raw material). All the procedure was repeated for five

times as discussed above. From the linear regression percentage recovery of Atisine was

determined.



Precision


experiment of the proposed method. Repeatability was evaluated by preparing and

analyzing the standard solution of the drug six times. The intra day reproducibility was

determined by analyzing freshly prepared solution in triplicate at three different

concentration whereas inter day reproducibility was checked by analyzing the standard

solutions at six different days under same operative condition.

Limit of Detection and Limit of Quantification

Limit of detection and Quantification of atisine was calculated visually by error and trial.

Specificity


specifically in the presence of component that may be expected to be present in the

sample matrix. 30 µg of test and standard atisine were spotted on the TLC plate,

developed and scanned as described above. The test chromatogram was compared with

the standard.




4.3.1 Pharmacognostic and physicochemical evaluation

4.3.1.1 Microscopical characters of the Kurchi and Ativish powder4

Study confirms the presence of all the identifying characters of both the drugs in all the

samples. The microscopical characters of the kurchi bark powder and ativish root are

mentioned Figure 4.1 and Figure 4.2 respectively.

1 2 3

Figure 4.1 Microscopic characters of kurchi bark powder

1 Cork cell

2 Cork cell with Starch grain

3 Fibrvascular tissue

1 2 3

Figure 4.2 Microscopic characters of Ativish root powder

1 Xylem vessel

2 Cork cell

3 Starch grains



4.3.1.2 Foreign matter

All the samples both the drugs have shown minimum amount of foreign matter which is

within the pharmacopoeial limit. Amount of foreign matter in both the drugs is mentioned

in Table 4.1.

4.3.1.3 Extractive values

The results of water soluble extractives and alcohol soluble extractives of all the samples

of Kurchi and Ativish powder are mentioned in Table 4.1. High values of these indicate

the presence of good amount of water and alcohol soluble chemical constituents of both

the drugs. Both the drugs have alkaloids which are believed to be responsible for the said

therapeutic activities of the drugs may enrich in these extracts.

4.3.1.4 Ash values

Results of the experiment on the ash values of all the samples of both the drugs are given

in Table 4.1. It appears from the results that both Kurchi and Ativish have all the results

in agreement with those mentioned in pharmacopoeia. However, both the drugs have

mainly water soluble ash and very low amount of acid insoluble ash suggesting the

acceptable range of undesired heavy metal impurities.

Table 4.1: Foreign matter, extractives and ash value of Kurchi and Ativish

Raw material Foreign

matter

(% w/w)

Ash value (% w/w) Extractives (% w/w)

Total Acid

insoluble

Water

soluble

Water

soluble

Ethanol

soluble

Kurchi bark-1 0.14 5.20 0.37 96.60 21.54 19.58

Kurchi bark-2 0.24 4.80 0.42 91.25 22.41 20.14

Kurchi powder 0.20 5.10 0.39 92.35 19.87 21.07

Ativish root-1 0.13 3.60 0.60 83.33 23.87 25.67

Ativish root-2 0.09 3.58 0.58 83.33 23.56 26.12

Ativish powder 0.10 2.98 0.47 84.22 22.13 24.96



4.3.2 Total alkaloids of kurchi and Ativish2,3

The results of the analysis of the all the samples of both the drug suggest the suitability of

respective I.P methods. These methods seem to be suitable for the proximate analysis of

total alkaloids in the respective drugs. All the samples of Kurchi bark and Ativish root

contained nearly 4.0 % w/w of total alkaloids, which is quite in agreement with the

reported yield in the literatures (Figure 4.2).

Table 4.2: Total alkaloids of Kurchi and Ativish

Raw material Kurchi

and Ativish

Total alkaloids (% w/w)

Kurchi bark-1 3.76

Kurchi bark-2 4.05

Kurchi powder 3.84

Ativish root-1 4.19

Ativish root-2 4.37

Ativish powder 4.08

4.3.3 Estimation of conessine in Kurchi by HPTLC4

4.3.3.1 Chromatogram

Conessine formed a reddish brown zone on a white background with an RF of 0.73 ±

0.0051 after development and detection as described method (Figure 4.3). Because the

conessine did not show absorbance when viewed under UV light, selective detection in

visible mode after spraying with dragondroff and then sodium nitrite reagent was carried

out. The mobile phase comprised of Toluene: Ethyl acetate: Diethyl amine (6.5:2.5:1)

was gave better resolution of all the compounds among the mobile phase tried.



1 2 3 4 5 6 7 8

Figure 4.3 Photograph of a TLC plate showing separation of conessine and other

constituents from the standard (Tracks 1–5) and test solutions (Tracks 6-8).

4.3.3.2 Assay of conessine in Kurchi

Results of the analysis of the different samples of kurchi by proposed HPTLC method is

given in Table 4.3. There is no noticeable difference found in the amount of conessine

among the samples of kurchi which were procured from different suppliers. However the

samples of the intact bark of kurchi show little more amount of conessine than the

powdered samples of kurchi bark. Kurchi bark-1 and kurchi bark-2 represents the intact

raw material, whereas kurchi powder was procured directly from the ayurvedic drug

supplier.

Table 4.3: Assay of conessine in different samples of Kurchi bark by HPTLC

Raw material Theoretical amount

of conessine (%w/w)

Amount of conessine

found ± S.D (% w/w)

(n=3)

Kurchi bark-1

Kurchi bark-2

Kurchi powder

0.4

0.4

0.4

0.373 ± 0.023

0.386 ± 0.033

0.355 ± 0.022



4.3.3.3 Validation of proposed method5

Linearity and range

Linear correlation was obtained between peak areas and concentrations of conessine in

the range of 10-60 ng/spot. Characteristic parameters for regression equation and

correlation are given in Table 4.4. The linearity of the calibration graphs was validated by

the high value of correlation coefficients of the regression (Figure 4.4).

Table 4.4 Regression parameters for the analysis of conessine by HPTLC method.

Parameter

Value

Range

Slope

Intercept

Regression coefficient

Regression Equation

10-60 (ng/spot)

104.77

158.03

0.9942

Y = 104.77x + 158.03

Figure 4.4 Calibration curve of conessine by HPTLC method.

S T A N D A R D C U R V E O F C O N E S S I N E

y = 1 0 4 . 7 7 x + 1 5 8 . 0 3

R 2 = 0 . 9 9 4 2

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

0 2 0 4 0 6 0 8 0

C O N C E N T R A T I O N ( n g / s p o t )

AR

EA

(S

q.m

m)



Accuracy (% Recovery)

The recovery experiments were carried out as in the text. The percent recovery obtained

was 98.34 to 100.25%. The results of recovery study are given in Table 4.5.

Table 4.5 Data of recovery study of conessine by HPTLC method.

Theoretical amount

of conessine

(ng/spot)

Amount of

conessine

added (ng/spot)

Amount of

conessine

found (ng/spot)

% Recovery ± S.D.

(n=5)

9.6

9.6

9.6

10

15

20

19.65

24.33

29.11

100.25 ± 1.69

98.90 ± 1.27

98.34 ± 0.99

Precision

Method precision

Relative standard deviation of all the parameters is less than 2 % for the degree of

repeatability indicating the high repeatability of the proposed method.

Table 4.6 Method precision data of analysis of conessine by HPTLC.

Conessine

(30 ng/spot)

Rf value Peak area

1

2

3

4

5

6

Mean

SD

RSD (% CV)

0.73

0.73

0.72

0.73

0.73

0.73

0.7283

0.0051

0.56052

3340

3286

3321

3378

3401

3412

3356.333

49.07211

1.461162



Intermediate Precision

It was determined as in the text. Low value of % CV of intra-day (0.45-1.43) and inter-

day (1.06-2.02) precision reveal that the proposed method is precise (Table 4.7 & 4.8).

Table 4.7 Intra-day precision data of analysis of conessine by HPTLC method

Conessine

(ng/spot)

Mean ± S.D. (n=3) % C.V

10

20

30

40

50

60

1127.7 ± 12.44

2228.4 ± 31.97

3340.0 ± 37.43

4428.7 ± 47.55

5610.0 ± 29.50

6214.8 ± 28.19

1.103

1.434

1.104

1.070

0.528

0.453

Table 4.8 Inter-day precision data of analysis of conessine by HPTLC method

Conessine

(ng/spot)

Mean ± S.D. (n=6) % C.V

10

20

30

40

50

60

1137 ± 22.79

2314 ± 29.18

3401 ± 36.24

4521 ± 56.57

5715 ± 85.70

6310 ± 81.78

2.009

1.266

1.065

1.257

1.499

1.292

Limit of detection (LOD)

The limit of detection of the drug was calculated practically. LOD for conessine was

found to be 3 ng/spot.

Limit of quantification (LOQ)

The limit of quantification of the drug was calculated as practically. LOQ for Conessine

was found to be 10 ng/spot.



Specificity

Comparison of chromatogram of conessine from the test drug (Kurchi bark) and with

standard conessine, showed that conessine was separated from interference by the other

constituents and impurity if present (Figure 4.5and 4.6).

Figure 4.5 Chromatogram of connesine (30 ng/Spot), Peak: conessine: Rf: 0.73.

Figure 4.6 Chromatogram of Kurchi bark (30 ng/Spot), Peak: conessine: Rf: 0.73



4.3.3.4 Summary of validation parameter (Table 4.9)

Table 4.9 Summary of validation parameters of conessine by HPTLC

Parameter

Result

Linearity range (ng/spot)

Correlation coefficient

Precision (%CV)

Intra day (n=3)

Inter day (n=6)

Repeatability of sample application (n=6)

Repeatability of peak area (n=7)

%Recovery (n=5)

Limit of detection (ng/spot)

Limit of quantification (ng/spot)

Specificity

10-60

0.9942

0.45-1.43

1.065-2.02

0.56

1.46

98.34-100.25

3

10

Specific

4.3.4 Estimation of atisine in Ativish by HPTLC method4.

4.3.4.1 Chromatogram

Atisine shows maximum absorption at 274 nm (Figure 4.7) and also formed a reddish

brown zone on a white background with an RF of 0.39 ± 0.0051 for both the wavelength

after development and detection as described above (Figure 4.8). As the compound did not

show absorbance when viewed under UV light, selective detection reagent such as

dragondroff and then sodium nitrite were sprayed to visualize. Atisine shows the

absorption after spraying the dragondroff, but scanning the zones in visible absorbance

mode provided better quantitative results than UV absorbance scanning. The mobile

phase Toluene: Ethyl acetate: Diethyl amine (7:2:1) was used for the sepeation, but the

suggested detection reagent only dragondroff did not produce stable colored zones with a

light background color that would enable densitometric quantitative analysis.



Figure 4.7 UV Spectra of atisine show maximum absorption at 274 nm.

1 2 3 4 5 6

Figure 4.8 Photograph of the plate showing spots of atisine from standard solutions



4.3.4.2 Assay of atisine in Ativish

Results of the analysis of the different samples of Ativish by proposed HPTLC method is

given in Table 4.10. All the samples of Ativish shows little more amount of atisine when

detected at 520 nm than at 274 nm. Considerable difference was found in the amount of

atisine between the sample of Ativish root and Ativish powder. Ativish powder procured

from the local market show lower amount of atisine than the intact root. This might be

due to the improper collection of the raw material or long time storage after the

powdering of the material.

Table 4.10 Assay of atisine in different samples of Ativish by HPTLC

Raw material Theoretical amount

of atisine (%w/w)

Amount of atisine

found (% w/w)

(n=3)

At 520 nm At 274 nm

Ativish root-1

Ativish root-2

Ativish powder

0.4

0.4

0.4

3.53

3.86

3.23

3.46

3.75

3.19

4.3.4.3 Validation of proposed method5

Linearity and range

Linear correlation was obtained between peak areas and concentrations of atisine in

concentration range of 10-60 ng/spot. Characteristic parameters for regression equation

and correlation are given in Table 4.11.The linearity of the calibration graphs was

validated by the high value of correlation coefficients of the regression (Figure 4.9 &

4.10).



Table 4.11 Parameters of regression for the analysis of atisine by HPTLC method.

parameter Value

Slope

Intercept

Regression

coefficient

Regression

Equation

520 nm 274 nm

91.465

417.74

0.9974

Y = 91.465x

+ 417.74

33.661

419.2

0.9981

Y = 33.661x

+ 419.2

C a l i b r a t i o n c u r v e o f a t i s i n e a t 5 2 0

n m

y = 9 1 . 4 6 5 x + 4 1 7 . 7 4

R 2 = 0 . 9 9 7 4

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

0 2 0 4 0 6 0 8 0


AR

EA

(S

q m

m)

Figure 4.9 Calibration curve of atisine by HPTLC method at 520 nm.

C a l i b r a t i o n c u r v e o f a t i s i n e a t 2 7 4 n m

y = 3 3 . 6 6 1 x + 4 1 9 . 2

R2

= 0 . 9 9 8 1

01 0 0 02 0 0 03 0 0 0

0 2 0 4 0 6 0 8 0


AR

EA

(S

q m

m)

Figure 4.10 Calibration curve for analysis of atisine by HPTLC method at 274 nm.




The recovery experiments were carried out as in the text. The percent recoveries obtained

at 520 and 274 nm were 98.53 to 99.52 and 98.76 to 99.93 respectively. The results of

recovery study are given in Table 4.12.

Table 4.12 Data of recovery study of analysis of atisine by HPTLC method

Theoretical

amount

of drug

taken (ng/spot)

Amount of

drug

added

(ng/spot)

Amount of

drug found (ng/spot)

% Recovery ± S.D.

(n=5)

520 nm 274 nm 520 nm 274 nm

9.6

9.6

9.6

10

15

20

19.41

24.24

29.46

19.47

24.18

29.58

99.03 ±1.69

98.53 ± 1.27

99.52 ± 0.99

99.33±1.59

98.76 ±

1.17

99.93±1.01

Precision

Method precision

Relative standard deviation of all the parameters is less than 2% and 1% for degree of

repeatability of spotting (1.75 and 0.005%, 0.71 and 0.005%) respectively for 520 and

274 nm, which indicates that the proposed method is repeatable (Table 4.13)

Table 4.13 Method precision data for the analysis of atisine by HPTLC method

Atisine (30

ng/spot)

Rf value

Peak area

520 nm 274 nm 520 nm 274 nm

1

2

3

4

5

6

Mean

SD

RSD (%CV)

0.39

0.39

0.39

0.38

0.38

0.39

0.3866

0.0051

1.315511

0.39

0.39

0.39

0.38

0.38

0.39

0.3866

0.0051

1.335511

3280.0

3268.5

3301.5

3258.4

3242.1

3241.0

3265.25

23.29418

0.713397

1462.7

1437.5

1494.2

1435.7

1458.2

1425.1

1452.233

25.03195

1.7565




It was determined as in the text. The low % CV values of intra-day (0.59-1.53%, 0.81-

1.86%) and inter-day (0.67-2.075%, 1.19-2.94%) for 520 and 274 nm respectively reveal

that the proposed method is precise (Table 4.14 and 4.15).

Table 4.14 Intra-day precision data for analysis of atisine by HPTLC method.

Atisine (ng/spot) Mean ± S.D. (n=3) % C.V

274 nm

520 nm 274 nm 520 nm

10

20

30

40

50

60

725.8 ± 11.74

1090.3 ± 17.23

1472.7 ± 27.43

1785.1 ± 14.55

2091.0 ± 15.50

2419.2 ± 36.19

1269.9 ± 19.54

2279.5 ± 23.15

3280.0 ± 30.75

4052.6 ± 23.98

4866.0 ± 34.57

5966.0 ± 68.41

1.61

1.58

1.86

0.81

0.74

1.49

1.53

1.01

0.93

0.59

0.71

1.14

Table 4.15 Inter-day precision data for analysis of atisine by HPTLC method.

Atisine (ng/spot) Mean ± S.D. (n=6) % C.V

274 nm 520 nm 274 nm 520 nm

10

20

30

40

50

60

736 ± 21.64

1104.1 ± 21.18

1498.5 ± 26.56

1809.8 ± 29.41

2114.2 ± 25.24

2467.5 ± 58.45

1284.5 ± 26.64

2265.4 ± 21.24

3245.1 ± 25.31

4084.1 ± 27.54

4805.1 ± 38.77

6001.7 ± 84.85

2.94

1.91

1.77

1.62

1.19

2.36

2.07

0.93

0.77

0.67

0.80

1.41


The limit of detection of the drug was calculated practically. LOD for Atisine was found

to be 3 and 3.5 ng/spot for 520 and 274 nm respectively.




The limit of quantification of the drug was calculated as practically. LOQ for Atisine was

found to be 10 ng/spot for 520 and 274 nm.

Specificity

Comparison of chromatogram of atisine in ativish with reference atisine, showed no

interference from the other constituents and impurities. (Figure 4.11 and 4.12, 4.13)

Figure 4.11 Chromatogram of atisine in Ativish (30 ng/Spot), peak: atisine: Rf: 0.39.

Figure 4.12 Chromatogram of atisine standard at 274 nm (30 ng/Spot), peak:

atisine: Rf: 0.39.



Figure 4.13 Chromatogram of atisine standard at 520 nm (30 ng/Spot), peak:

atisine: Rf: 0.39

4.3.4.4 Summary of validation parameters of the analysis of atisine by HPTLC

(Table 4.16)

Table 4.16 Summary of validation parameter of atisine by HPTLC

Parameter Result

520 nm 274 nm


Correlation co efficient

Precision (%CV)

Intra day (n=3)

Inter day (n=6)

Repeatability of sample

application (n=6)

Repeatability of peak area

(n=7)

%Recovery (n=5)

Limit of detection (ng/spot)

Limit of quantification

(ng/spot)

Specificity

10-60

0.9974

0.59-1.53

0.67-2.07

1.33

0.71

98.47-99.51

3

10

Specific

10-60

0.9981

0.74-1.86

1.19-2.94

1.33

1.75

9919-99.93

3.5

10

Specific



4.4 References

1. “Quality control methods for medicinal plant materials” by WHO Geneva, 2002.



4. “Quality standards of indian medicinal plants” by ICDR, New Delhi, Vol-I, 109-116.

5. ICH Guidelines “Validation of analytical methodology”.

6. Plant drug analysis by H. Wagner and S. Bladt, Springer, 4th

Edition, 360.

Chapter-5 Evaluation of Kutaj Ghanvati

S.K.P.C.P.E.R (M Pharm Dissertation) 81 Arun M Prajapati

Chapter 5

Evaluation of Kutaj Ghanvati

5.1 Introduction

Efficacy and safety of the herbal drugs and formulations including ayurvedic and other

traditional formulations are always under a big question mark. These are mainly

concerned with the quality of raw material and methodology adopted during the

procurement, handling and manufacturing them. The prime importance is given to the

standardization of the formulations which are used in disease to ascertain the efficacy and

safety. Many guidelines are published especially by WHO, Indian Herbal

Pharmacopoeia, Ayurvedic formulary and many others for the standardization and

evaluation of these materials.

Hence, in the present investigation the attempt was made to evaluate the Kutaj Ghanvati

formulation under the study for the pharmacognostic and physicochemical parameters

according to these guidelines. Also, the detailed methodology is also described to find out

the amount of active constituents present in these formulations. Also they were evaluated

by pharmacotechnical parameter for tablet (Ghanvati).



5.2 Experimental

5.2.1 Material

Kutaj ghanvati is well known ayurvedic formulation, which contain Kurchi bark and

Ativish root extract. Different market formulations were collected from three well known

ayurvedic pharmacy and one sample formulation of Kutaj Ghanvati was prepared

according to the Siddha yog sangraha from the authentic raw material in the laboratory.

A, B, C are the formulations collected from the market and L is the formulation prepared

in the laboratory. All the market formulations contained 8 part of Kurchi extract and 1

part of Ativish extract. All the tablets were crushed with the use of glass mortar and

pastel and from that required quantity of powder were taken and used.

5.2.1 Preparation of Kutaj Ghanvati1.

Method of preparation of kutaj ghanvati is given in Siddha yog sangraha. 10 gm of kurchi

bark powder (60mesh) was accurately weighed and extracted with 100 ml of distilled

water on a burner till the volume was reduce up to 50, cooled then filtered through

muslin. The mass pressed and rinsed with fresh two 10 ml quantities of distilled water.

This extract was used to make 20 pills. Each pill representing extract of 0.5 gm Kurchi

powder. The aqueous extract further concentrated to a syrup liquid, to this 7 gm of

powder (60 mesh) of Ativish was added to make mass of 20 pills. So that each pills

representing 0.357 gm of Ativish root powder. The total mass of pills was weighed and

from that 20 pills were prepared. Each pills representing 0.0.450 gm of weight After

drying the pills, which were contained 9 gm of 20 pills. Each pill representing 0.450 gm

of weight.

5.2.2 Pharmacognostic and physicochemical evaluation of Kutaj Ghanvati2

5.2.2.1 Microscopic examination2

Since, the material is powdered sample macroscopical study for formulation was skipped

off. Powder characteristics of the Ativish were studied under the microscope. The stained

and unstained slide was prepared and the characters were examined and photographed

using CCD camera.



Method

1) About 1-2 gm powders was taken and dissolved in methanol, shaken for few minutes

and then it was filtered. The filtrate which contain extractable matter and chemical,

while the impurities and powder material were remained on the filter paper. Residue

was boiled with chloral hydrate foe few minutes. After boiling the power it was stained

with the HCL and phloroglucinol and at last washed with water. The powder was

mounted on the slide with lactophenol and covered with the cover slip. The slide was

examined under the microscope.

2) Unstained slide was also prepared as above and examined.

3) The iodine stained slide was also prepared and examined under the microscope.

5.2.2.2 Determination of Ash Value2

Total ash

4 g of the formulation powder was taken, accurately weighed, in a previously changed

and tared crucible (silica). The material was placed in an even layer and ignite it by

gradually increasing the heat to 500-600 0C until it is white, indicating the absence of

carbon. Cooled in a dessicator and weighed, moistened the residue with about 2 ml of

ammonium nitrate R. Dried on a water-bath, then on a hot plate and ignite to constant

weight. Allowed the residue to cool in a suitable dessicator for 30 minutes, and then

weighed without delay. % of total ash was determined with respect to dry wt of drug.

Acid-insoluble ash

To the crucible containing the total ash, 25 ml of HCL (~70g/l) was added, covered with

a watch glass and boiled gently for 5 minutes. The watch glass was rinsed with 5ml of hot

water and washing was added to the crucible. Insoluble matter was collected on an ash

less filter paper and washing of this filter paper was carried out with hot water until the

filtrate was remaining neutral. The filter paper containing the insoluble matter was

transferred to the original crucible, which is then dried on a hot plate and ignited to

constant weight. Allowed the residue to cool in a suitable desiccator for 30 minutes, and

then weighed without delay. The content of acid-insoluble ash was calculated with

respect to the weight of air dried powdered plant material.



Water-soluble ash

To the crucible containing total ash, 25 ml of water was added and boiled for 5 minutes.

Insoluble matter was collected on an ash less filter paper. The residue was washed with

hot water and ignited in a crucible for 15 minutes at a temperature not exceeding 450 0C.

The weight of the residue was substracted from the weight of total ash. The content of

water soluble ash was determined with respect to the weight of the air dried powdered

plant material.

5.2.3 Determination of total alkaloids of Kurchi in Kutaj Ghanvati3

Estimation of total alkaloids of Kurchi in Kutaj Ghanvati as mentioned earlier “Kutaj

Ghanvati” should contain 8 part of powder extract, in which water was a efficient

menstrum for measurement of alkaloids. To judge this, analysis of prepared and marketed

product were carried out as follow.

20 pills were weighed and average weight of each pill was determined. 20 pills were

powdered in a mortar and weighed accurately powdered mass representing 10 such pills

equivalent to 5 gm of powdered kurchi bark. The 5 gm powder was transferred in conical

flask and dissolved in the 50 0.1 M HCL solution.

This acid extract was made alkaline with strong solution of ammonia extracted with four

10 ml portion of solution of chloroform for three times. Each chloroform was washed

with same 10 ml portion of water continued in different separator. Combined the

chloroform extracts and added 20 ml of N/10 sulphuric acid and shaken well for 5

minutes. Chloroform was washed with two 20 ml portion of N/20 H2SO4. Excess acid

was titrated using 0.1 N NaOH using Phenol Red, which show color change from red to

light green at the end of the titration. Total alkaloid was estimated using the factor given

in the IP.

Factor: Each ml of N/10 H2SO4is equivalent to 0.01657 go f total alkaloids of kurchi.



5.2.4 Estimation of conessine and atisine in kutaj Ghanvati by HPTLC4.

5.2.4.1 Material and chemicals

Kutaj Ghanvati

Kurchi Powder

Conessine Standard

Atisine Standard

Ativish powder

Ammonia

Diethyl ether

Methanol

Chloroform

Toluene

Ethyl acetate

Diethyl amine

Dragon-droff’s reagent


Distilled water

Spraying bottle

Glass mortal and pastel

Conessine and atisine reference standard were obtained as gift samples from Cadila

pharmaceutical LTD, Dholka.









5.2.4.3 Chromatographic condition



Mobile phase: Toluene: Ethyl acetate: Diethyl amine (4:5:1)


Band width: 6 mm



Distance run: 80mm

Spraying reagent: Dragon-droff’s was sprayed after drying the plate and then



Scanning Wave length : 520nm



Temperature: 25 0C


Conessine and Atisine stock standard solution was prepared by weighing and diluting 40

and 10 mg of standard Conessine and Atisine up to 50ml and 100 ml with absolute

methanol. 1 ml solution of both were taken and diluted up to 10 ml with methanol.


20 tablets were weighed and powdered with glass mortal and pastel then 1 gm powder of

kutaj ghanvati was taken extracted with 40 ml of methanol.. Filtered, evaporated and

dissolved the residue in 40 ml methanol and then diluted up to 100 ml with methanol. 1

ml from this was taken and diluted up to 10 ml with methanol.







TLC standard solution of conessine and atisine (80-400ng and 10-50ng), both the

solution were over spotted by Linomat-V spotter. The plate was developed in the mobile

phase specified in the chromatographic condition. Conessine and Atisine were detected

by spraying lightly and evenly (not to wetness) with Dragon-droff’s and dried at room

temperature in the air. After drying the plate Sodium nitrite was sprayed on it and again

dried in air for minimum of 20 minutes and then Standard zones were scanned at 520 nm

with Scanner-3 as mentioned the chromatographic condition above.

5.2.4.7 Estimation of conessine and atisine in raw material

From both the sample solutions 2.5, 3.5 and 4.5µl were over spotted on the precoated

silica gel plate and process was repeated to develop and scan the plate as mentioned

above. A calibration equation relating to the standard Concentration to scan areas was

determined by use of a linear regression program on a personal computer, and the weight

of Atisine in the sample was calculated from the calibration equation by using the

average area of triplicate sample aliquots. The experimental weight was compared with

the theoretical label value.



Detection, Limit of Quantitation, Accuracy and Specificity.

Linearity

Linearity of the method was performed by analyzing standard solution of Conessine and

Atisine by the proposed method in concentration range 80-400 nm and 10 to 60 ng/spot.

Accuracy

Accuracy of the proposed method was determined by recovery study. Recovery studies

were carried out by adding three different quantities of conessine and atisine were over

spotted (80, 120, and 160 ng/ml and 10, 15 and 20 ng/ml) to preanalyzed solution of

Sample (Kutaj Ghanvati). All the procedure was repeated for five times as discussed

above. From the linear regression percentage recovery of conessine and atisine were

determined.



Precision


experiment of the proposed method. Repeatability was evaluated by preparing and

analyzing the standard solution of the drug six times. The intra day reproducibility was

determined by analyzing freshly prepared solution in triplicate at three different

concentration whereas inter day reproducibility was checked by analyzing the standard

solutions at six different days under same operative condition.

Limit of Detection and Limit of Quantitation

Limit of detection and Quantitation of both drugs were calculated visually by trial and

error.

Specificity


specifically in the presence of component that may be expected to be present in the

sample matrix. 240 and 30 ng of test and standard atisine were spotted on the TLC plate,

developed and scanned as described above. The test chromatogram was compared with

the standard.

5.2.5 Evaluation of tablet parameter of Kutaj Ghanvati

All the samples of Kutaj Ghanvati were subjected to the series of tests such as friability,

hardness, disintegration test, dissolution studies and assay of Ghanvati (tablet).

Friability7

The friability of the tablets was measured in a Roche friabilator (Camp-bell Electronics,

Mumbai). Tablets of a known weight (W0) or a sample of 10 tablets are dedusted in a

drum for a fixed time (100 revolutions) and weighed (W) again. Percentage friability was

calculated from the loss in weight as given in equation as below. The weight loss should

not be more than 1 %.

% Friability = (W0 W)/W0 100 ---------------- (c)



Hardness9

The hardness of the tablets was determined by diametric compression using a dial type

hardness tester (Model no 1101, Shivani Scientific Ind). A tablet hardness of about 4-5 kg

is considered adequate for mechanical stability. Determinations were made in triplicate.

Disintegration test8

The disintegration time (DT) of the tablets was determined in distilled water at 37 ± 0.5o

C using disintegration test apparatus (Electrolab ED-2 Bowl USP, Mumbai). One tablet

was placed in each of the 6 tubes of the basket and the time taken for all the tablets to

disintegrate and go through the wire mesh was recorded. The disintegration time should

not be more than 15 minutes. Determination test was carried out in triplicate.

Dissolution study10

The drug release study was carried out using USP XXIII paddle apparatus (Veego VDA –

8D) at 37 ± 0.5o C and 50 rpm using 900 ml of 0.1 N HCL as a dissolution medium for a

period of two hour. As Kutaj Ghanvati (tablet) contains alkaloidal principles, HCL is the

ideal medium for dissolution. Sixty ml of sample was withdrawn at predetermined the

end of two hour study, filtered through a Whatman filter paper. The volume of the filtrate

was adjusted to 60 ml and basified with dilute ammonia solution and extracted thrice with

chloroform. The combined chloroform was evaporated in water bath and the residue was

reconstituted in 60 ml of 0.2 N H2SO4. The content of total alkaloids was determined by

titrimetric assay following IP 1955 method. Studies were carried out in triplicates for all

the samples of Kutaj Ghanvati.




5.3.1 Pharmacognostic and physicochemical evaluation of Kutaj Ghanvati

5.3.1.1 Microscopical examination of Kutaj Ghanvati

The results of the microscopical evaluation are quite interesting. Only laboratory sample

of Kutaj Ghanvati shows the presence of powder characters of the Ativish root. As

Kurchi bark extract is incorporated in this sample, no characters of kurchi were found.

All the samples of marketed formulation of Kutaj Ghanvati have utilized extract of the

ingredients, no powder characters were observed. Figure 5.1 shows the microscopical

characters of Ativish in Kutaj Ghanvati.

1 2 3

Figure 5.1 Microscopic characters of ativish in Kutaj Ghanvati

1 Xylem vessel

2 Cork cell

3 Starch grain

5.3.1.2 Ash values of Kutaj Ghanvati

The results in Table 5.1 reveal that laboratory sample contained very less amount of total

ash, where as market samples shows comparatively higher amount of total ash. The

reason behind this may be the incorporation of mineral diluents in the formulation for the

tablet form of the Ghanvati



Table 5.1 Ash values of samples of Kutaj Ghanvati

Formulation Ash value (%)

Total ash

value

Acid

insoluble

Ash

Water

soluble

Ash

A 5.40 0.21 96.29

B 6.20 0.35 95.16

C 4.89 0.37 92.43

L 0.65 0.25 96.66

5.3.2 Total alkaloids of Kutaj Ghanvati3

Results of the analysis in Table 5.2 indicate that all the marketed samples of the Kutaj

Ghanvati contains nearly equal amounts of total alkaloids with respect to the weight of

the tablet (11.27 % to 13.12 %). However, laboratory sample gave only 6.66 % of total

alkaloids. The laboratory sample was prepared according to the principles of Ayurveda.

The marketed sample of the Kutaj Ghanvati, which are prepared from the extract of both

the ingredients drugs obviously contained higher amount of alkaloids. Alkaloids of

Ativish are considered poisonous, hence at what extent the incorporation of the extract

form of Ativish is acceptable is the matter of question mark. Also, it is not sure that

which varieties of Ativish (Aconitum) are used for the preparation of extract. Aconitum

heterophyllum is the genuine drug mentioned in Ayurveda as Ativish and it does not

contain a poisonous principle- aconitine which is found present in other varieties of

Aconitum such as Aconitum nepellus.

Table 5.2 Assay of Kutaj Ghanvati for the total alkaloids

Formulation Weight of

Ghanvati

(gm) ± S.D

Total alkaloids

/Ghanvati

(gm) ± S.D

Percent alkaloid

with respect to

wt. of Ghanvati

Theoritcal

content of total

alkaloids

A

B

C

L

0.754 ± 0.006

0.650±0.004

0.327±0.008

0.450±0.007

0.085±0.004

0.074±0.003

0.042±0.001

0.030±0.002

11.27

11.38

13.12

6.66

-

-

-

0.032



5.3.3 Simultaneous estimation of conessine ant atisine in Kutaj Ghanvati by HPTLC

5.3.3.1 Chromatogram of Kutaj Ghanvati by HPTLC

Conessine and Atisine formed a reddish brown zone on a white background with an RF of

0.36 ± 0.0040 and 0.72 ± 0.0040 after development and detection as described above

(Figure 5.2 & 5.3). Because the compound conessine does not show absorbance when

viewed under UV light, and has no functional group enabling use of a selective detection

reagent such as dragon-droff’s and then sodium nitrite. Conessine and Atisine both show

the absorption after spraying the dragon-droff’s and then Sodium nitrite6, scanning the

zones in visible absorbance mode provided better quantitative results than UV

absorbance scanning, The mobile phase Toluene: Ethyl acetate: Diethyl amine (4:5:1)

was used for the analyses, the suggested detection reagents produce stable colored zones

with a light background color that would able densitometry quantitative analysis.

1 2 3 4 5 6 7 8

Figure 5.2 Photograph of a plate containing chromatograms obtained from

standard solutions of conessine and atisine (tracks 1–8).



1 2 3

Figure 5.3 Photograph of a plate containing chromatograms obtained from

formulation of conessine and atisine (Tracks 1–3).

5.3.3.2 Assay of conessine and atisine in Kutaj Ghanvati

Results of the analysis of the different samples of Kutaj Ghanvati by proposed HPTLC

method is given in Table 5.3. It appear from the table that market sample which are

prepared from the extract of both the drugs contain nearly equal amount of conessine and

atisine.

Table 5.3 Assay of conessine and atisine in different samples of Kurchi bark by

HPTLC

Formulation Weight of

Ghanvati

(gm) ± S.D

Conessine /Ghanvati

(mg) ± S.D

Percent alkaloid

with respect

to wt. of Ghanvati

Conessine Atisine Conessine Atisine

A

B

C

L

0.754±0.006

0.650±0.004

0.327±0.008

0.450±0.007

3.26±0.002

2.67±0.001

1.32±0.002

2.00±0.002

0.40±0.001

0.34±0.001

0.16±0.001

0.25±0.002

0.71

0.58

0.29

0.44

0.035

0.041

0.036

0.025



5.3.3.3 Validation of HPTLC method 5

Linearity and range

Linear correlation was obtained between peak areas and concentrations of conessine and

atisine in concentration range of 80-400 and 10-50 ng/spot. Characteristic parameters for

regression equation and correlation are given in (Table 5.4). The linearity of the

calibration graphs was validated by the high value of correlation coefficients of the

regression (Fig 5.4 & 5.5)

Table 5.4 Regression Parameter for analysis of conessine and atisine by HPTLC

method.

Parameter

Value

Conessine Atisine

Range

Slope

Intercept

Regression

coefficient

Regression

Equation

80-400 (ng/µl)

22.046

4652

0.9933

Y = 22.046x +

4652

10-60 (ng/µl)

85.381

615.11

0.9967

Y = 85.318x

+615.11

Figure 5.4 Calibration curve of analysis of atisine by HPTLC method.

STANDARD CURVE OF ATISINE y = 85.381x + 615.11

R2 = 0.9967

0

1000

2000

3000

4000

5000

6000

0 10 20 30 40 50 60

CONCENTRATION (ng/spot0

AR

EA

(S

q m

m)



STANDARD CURVE OF CONESSINE y = 22.046x + 4652

R2 = 0.9933

0

2000

4000

6000

8000

10000

12000

14000

16000

0 100 200 300 400 500

CONSENTRATION (ng/spot)

AR

EA

(S

q m

m)

Figure 5.5 Calibration curve of analysis of conessine by HPTLC method.


The recovery experiments were carried out as in the text. The percent recoveries obtained

were 98.31 - 100.01% and 98.30 – 100.72% respectively for Conessine and Atisine. The

results of recovery study are given in (Table 5.5)

Table 5.5 Recovery study of conessine and atisine in Kutaj Ghanvati by HPTLC

Theoretical amount of

drug taken (ng/spot)

Amount of drug

added (ng/spot)

Practical amount of

drug Found (ng/spot)

% Recovery ± S.D.

(n=5)

Conessine Atisine Conessine Atisine Conessine Atisine Conessine Atisine

A 68.05

B 62.48

C 57.64

L 66.34

A 68.05

B 62.48

C 57.64

L 66.34

A 68.05

B 62.48

C 57.64

L 66.34

7.62

7.13

6.88

8.60

7.62

7.13

6.88

8.60

7.62

7.13

6.88

8.60

80

80

80

80

120

120

120

120

160

160

160

160

10

10

10

10

15

15

15

15

20

20

20

20

148.23

142.49

137.59

144.82

185.92

182.5

176.55

183.2

225.0

221.58

215.61

224.41

17.49

17.04

16.90

18.41

22.37

21.78

21.88

23.42

27.82

26.67

26.44

28.49

99.92±0.86

100±1.67

99.96±1.02

98.96±0.99

98.86±0.84

100.01±1.48

99.38±0.97

98.31±0.84

98.66±1.03

99.59±.28

99.06±0.98

99.14±1.36

99.26±0.74

99.47±0.82

100.11±0.96

98.97±0.95

98.89±0.45

98.41±1.16

100.00±0.94

99.23±1.24

100.72±0.98

98.30±0.67

98.28±1.17

99.61±0.97



Precision

Method precision

Relative standard deviation is less than 2% and 1% (Table 5.6) for degree of repeatability

of spotting for Rf and Area were respectively for Conessine (0.568 and 1.266) and Atisine

(0.518 and 1.139)

Table 5.6 Method Precision data of analysis of conessine and atisine by HPTLC.

Reading Atisine (30ng/spot)

Conessine(240ng/spo)

Rf value Peak area Rf value Peak area

1

2

3

4

5

6

Mean

SD

RSD (% CV)

0.36

0.36

0.35

0.36

0.36

0.36

0.358333

0.004082

1.13928

3275.2

3251.8

3264.2

3258.4

3225

3258.1

3255.45

16.86935

0.5181

0.72

0.72

0.72

0.71

0.72

0.72

0.71833

0.004082

0.56832

9592.5

9875.4

9572.5

9554.1

9697.4

9594.1

9647.667

122.1911

1.2665


It was determined as in the text. The low % CV values of intra-day (0.87-1.96%, 0.67-

1.67%) and inter-day (0.07-2.98%, 0.92-2,02%) respectively for Conessine and Atisine,

precision reveal that the proposed method is precise (Table 5.7 and 5.8).



Table 5.7 Intra-day precision data of analysis of conessine and atisine by HPTLC

method

Concentration

(ng/spot)

Mean ± S.D. (n=3)

% C.V

Conessine Atisine Conessine Atisine Conessine Atisine

80

160

240

320

400

10

20

30

40

50

6687.1±25.42

7958.5±89.13

9687.5±94.93

11794.5±102.61

13870.4±271.88

1382±88.97

2387.3±24.82

3275.2±21.94

3987.0±45.85

4551.2±60.98

1.84

1.12

0.98

0.87

1.96

1.67

1.04

0.67

1.15

1.34

Table 5.8 Inter-day precision data of analysis of conessine and atisine by HPTLC

method

Concentration

(ng/spot)

Mean

± S.D. (n=6)

% C.V

Conessine Atisine Conessine Atisine Conessine Atisine

80

160

240

320

400

10

20

30

40

50

6704.8± 145.49

6967.5 ± 90.57

9712.6 ± 97.12

11804.3 ± 131.02

13883.1± 413.71

1375 ± 27.77

2359.5 ± 45.06

3267.2 ± 31.69

3974.1 ± 40.58

4843.7 ± 94.93

2.17

1.23

1.07

1.11

2.98

2.02

1.91

0.97

0.92

1.96


The limit of detection of the drug was calculated practically. LOD for conessine and

Atisine were found to be 3 ng/spot.


The limit of quantification of the drug was calculated as practically. LOQ for Conessine

and Atisine were found to be 10 ng/spot.



Specificity

Comparison of chromatogram of Conessine and Atisine in formulation (Figure 5.6 ) with

standard (Figure 5.7) Conessine and Atisine, showed no interference from the excipients

and impurity or any other adulterants.

Figure 5.6 Chromatogram of conessine and atisine formulation (30 and 240 ng/spot),

peak: conessine and atisine: Rf: 0.72 and 0.36

Figure 5.7 Chromatogram of conessine and atisine standard (30 and 240 ng/spot),

peak: conessine and atisine: Rf: 0.72 and 0.36



5.3.3.4 The summary of all validation parameters (Table 5.9)

Table 5.9 Summary of validation parameters of conessine and atisine by HPTLC

Parameter Result

Conessine Atisine


Correlation co efficient

Precision (%CV)

Intra day (n=3)

Inter day (n=6)

Repeatability of sample

application (n=6)

Repeatability of peak

area (n=7)

%Recovery (n=5)

Limit of detection

(ng/spot)

Limit of quantification

(ng/spot)

Specificity

80-400

0.9967

0.87-1.96

1.07-2.98

1.13

0.51

98.31 –100.01

3

10

Specific

10-50

0.9933

0.67-1.67

0.92-2.02

0.56

1.26

98.28-100.72

3

10

Specific



5.3.4 Evaluation of tablet parameters of Kutaj Ghanvati

5.3.4.1 Friability

Friability of laboratory sample of Kutaj Ghanvati is very high (1.67 %). Friability of

market samples of Kutaj Ghanvati is within the pharmacopoeial limit (0.12 % to 0.47 %).

The Ghanvati is not a compressed formulation and no binding agent was added in the

formulation. Also one of the ingredients (Ativish) is incorporated in the crude powder

form. These factor seems to be responsible for the high friability of Kutaj Ghanvati.

Whereas, the market formulations are in the form of compressed tablet. During the

manufacturing of compressed tablet generally binding agents are added to provide

firmness. These is why market samples show less friability (Table 5.10).

5.3.4.2 Crushing strength.

The crushing strength of all the formulation is more than sufficient. Generally

compressed tablet dosage form possess hardness of about 4 to 6 kg/cm2. Sample A and B

have shown very high crushing strength ( 10 and 8.5 kg/cm2) whereas, sample C and

laboratory sample have shown the crushing strength (5.2 and 4.8). The extract of the

drugs which are generally sticky and adhesive sufficiently contribute the hardness when

compressed (Table 5.10).

5.3.4.3 Disintegration

The results of the disintegratin test of all the samples are given in Table 5.10. It appears

from the results that disintegration time of Sample A is very high (37 min). Laboratory

sample shows less disintegratin time (15 min). It is clear from the reulsts that the

compressed tablet formulations of Kutaj Ghanvati have high disintegration time in

comparision with un compressed laboratory sample of Kutaj Ghanvati. It is very

interesting to conclude here that all the market samples fail disintegration test considering

the pharmacopoeal limit of DT is 15 minute.



Table 5.10 Friability, Disintegration and Crushing strength of Kutaj Ghanvati

Formulation % Friability Crushing strength

(kg/cm2)

Disintegration time

(min)

A

B

C

L

0.15

0.47

0.12

1.67

10.0

8.5

5.2

4.8

37

26

17

15

5.3.4.4 In vitro Dissolution study

The in vitro dissolution study of all the samples was carried out in 0.1 N HCL. After the

period of two hours the total alkaloidal content released in the medium was analyzed by

the method given in the experimental. Results of the test shows that not more than 30 %

of the total alkaloids are released in the medium. Sample C released only 2.7 % of total

alkaloids from the dosage form. Laboratory sample shows maximum release of total

alkaloids present in the formulation. It is very interesting that how these formulations of

Kutaj Ghanvati with this release pattern contribute their efficacy for the purpose they are

used (Table 5.11).

Table 5.11 % of total alkaloids of Kutaj Ghanvati released after 2 hour.

Formulation % of total alkaloids

released after 2 hour

A

B

C

L

21.62

15.54

2.70

30.25



5.4 References

1. Siddha Yog Sangraha, 8th

edition, 1984; 24.

2. “Quality control methods for medicinal plant materials” by WHO, Geneva, 2002.

3. Indian Pharmacopoeia, 1955; 358..

4. “Quality standards of indian medicinal plants” by ICDR, New Delhi, Vol-I, 109-116.

5. ICH Guidelines “Validation of analytical methodology”.

6. Plant drug analysis by H. Wagner and S. Bladt, Springer, 4th

Edition, 360.

7. Lachman.l, lieberman. A, kinig.j.l. The theory and practice of industrial pharmacy, 4th

edition, varghese publishing house, bombay.1991: 67-68

8. The united pharmacopoeia XXIV and national formulary 19. 2000 U.S.

pharmacopoeial convention: 2426.

9. J. R. R kurup, n. . T. Fell, j. M. Newton. J pharm. Sci. 1970; 59: 688–91.

10. T Pilpel. Asian j. Pharm. Sci. 1979; 1: 75–90.

Chapter-6 Conclusion


Chapter 6

Conclusion

Following conclusion have been drawn from the details study carried out under the aim

of pharmacognostical and pharmacotechnical evaluation of Kutaj Ghanvati.

The raw materials for the Kutaj Ghanvati, Kurchi bark and Ativish root are

procured from different location. It is concluded from the pharmacognostical and

physicochemical studies that all the raw materials are genuine. Even powdered

ingredients of Kurchi and Ativish are having same quality standards when

compared with the genuine intact form.

The total alkaloids of the raw materials when determined by the known

pharmacopoeial methods are in agreement with the theoretical amount present in

the genuine drugs. The result shows that Kurchi bark sample-1 and sample-2

contain 3.76 % w/w 4.0 5% w/w of total alkaloids. The powdered sample of

Kurchi bark contains 3.84 % w/w of total alkaloids. Ativish root sample-1 and

sample-2 contain 4.19 % w/w and 4.37 % w/w of total alkaloids. Powdered

sample of Ativish contains 4.08 % w/w.

The proposed HPTLC methods of analysis of raw material for the marker

compounds, conessine and atisine from the respective drugs- Kurchi and Ativish

seems to be accurate, precise, reproducible and repeatable. It is the first time,

when different samples of these drugs are estimated and compared for the

respective active constituents.

The proposed simultaneous estimation method of conessine and atisine from the

Kutaj Ghanvati is accurate, precise, reproducible and repeatable. The results of

the analysis of different samples of Kutaj Ghanvati gave very surprise results. The

amount of conessine and atisine in the different samples of Kutaj Ghanvati varies

from 0.29 % w/w to 0.71 % w/w and 0.025 % w/w to 0.041 % w/w respectively

with respect to the weight of Ghanvati (Tablet).

The high amount of total alkaloids and amount of conessine and atisine in market

samples may be attributed to the incorporation of concentrated extract of the

ingredient drugs.

Chapter-6 Conclusion


The results of the tablet parameters show that the market samples of Kutaj

Ghanvati, which are available as compressed tablet forms. They show high

crushing strength, low friability and delayed disintegration when compared with

the laboratory sample which was uncompressed and prepared according to the

concept of Ayurveda. It is also important to note that the release of the total

alkaloids from the Ghanvati in all the samples is very low (less than 30 %). The

market sample-C shows the least release of the total alkaloids 2.70% with respect

to the weight of Ghanvati.

Hence, it is very clear from this study that there is no uniformity in the process of

manufacturing of the Kutaj Ghanvati. There is a considerable difference in the results of

the parameters which are taken for the assessment of the Kutaj Ghanvati. It is essential to

focus these results and attempt must be made to extend the investigations for the

bioavailability studies and toxicological investigations of this formulation Kutaj Ghanvati

as it contain the toxic ingredients.

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