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©2015, Indian Pharmacopoeia Commission

All rights reserved

No part of this publication may be reproduced, stored in a

retrieval system/transmitted in any form by any means such

as electronic, mechanical including photocopying, recording

or otherwise without the prior permission of Indian Pharmacopoeia Commission.

ISBN:

Guidance Manual for Monographs Development

of Herbs and Herbal products.

On behalf of : Government of India

Ministry of Health and Family welfare

Produced and published by Indian Pharmacopoeia Commission

Government of India

Ministry of Health and Family Welfare

Sector 23, Raj Nagar,

Ghaziabad-201002

Tel: (91-120)- 2783401

Fax: (91-120)-2783401

Website: www.ipc.gov.in

Email: [email protected]

Designed and printed by:

Price: ₹ 250/-

Price : Inland ₹ 250/-

Foreign $

£

Disclaimer This manual is prepared as a guidance document for the stakeholders of IP who intend to submit Herbs and Herbal Products monographs,

candidate material for Phytochemicals Reference Substances and Botanical Reference Substances to IPC. They have been verified and

reviewed by experts before incorporation in this manual. Clarification on any matter presented in the manual will be issued with due

verification only. For any clarifications please consult IPC. The stakeholders are advised to keep abreast of the changes in the content if any,

from time to time through the technical secretariat of Indian Pharmacopoeia Commission. We also welcome suggestions for updating this

manual.

Copy right: The contents of this Manual are not to be copied in full or part or reproduced in any manner without the authority of the Indian

Pharmacopoeia Commission.

http://www.ipc.gov.in/

mailto:[email protected]


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OBJECTIVES

1. The objective of the “Guidance Manual for Monographs Development for Herbs and

Herbal Products” is to facilitate the concerned manufacturers and other stakeholders

for understanding the process and development of monographs of herbs and herbal

products.

2. The Manual also aims to encourage and promote research and development in the

quality of herbals in India and overseas.

3. The Manual also gives current Standard Operating Procedures (SOPs) for

development of monographs as well as Botanical Reference Substances (BRS) and

Phytochemicals Reference Substances (PRS) and invites suggestions for further

updating.

4. Indian Pharmacopoeia Commission will be glad to consider submission of data and

draft monographs adopting this Manual for inclusion in IP subject to such data

meeting the criteria laid down


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CONTENT

S. No. TITLE PAGE No.

Core Expert Committee

Acknowledgements

Abbreviations

4

6

7

1. Introduction 8

2. List of Monographs on Herbals in IP 2014 including those in Addendum 2015 10

3. Inclusion/ Exclusion Criteria of a Herbal Monograph in IP 11

4. Process of IP Herbal Monograph Development and Content of a Herbal

Monograph 13

5. General Monograph for Herbs, Processed Herbs and Herbal Products 20

6. Standard Operating Procedure for Preparation of Monographs of Herbs,

Processed Herbs and Herbal Products In IP

25

7. Standard Operating Procedure for Herbal Extracts Monographs 31

8. Standard Operating Procedure for Preparation, Qualification, Certification &

Supply of Botanicals Reference Substance (BRS)

35

9. Standard Operating Procedure for Preparation, Qualification, Certification &

Supply of Phytochemicals Reference Substance (PRS)

39

10. Identification of Herbal Materials 43

11. Determination of Ash 52

12. Determination of Extractive Values 54

13. Loss on Drying 56

14. Contaminants 57

15. Thin Layer Chromatography 60

16. High Performance Thin Layer Chromatography 66

17. High Performance Liquid Chromatography 74

18. Analysis of Essential Oils 82

19. Examples of Some Herbal Monographs 87

20. Herbal Drugs Manufacturers/Suppliers in India 93

21. Bibliography 95

22. Books for Further Reading on Herbs 96

23. Feedback Form 98


4

CORE EXPERT COMMITTEE

Chairman

Dr. G. N. Singh

Secretary-cum-Scientific Director,

Indian Pharmacopoeia Commission,

Ghaziabad

and

DCG (I),

Central Drugs Standard Control Organization (CDSCO),

FDA Bhawan, Kotla Road, New Delhi

Members

Sub-Group on Essential oils

Dr. D.B. Anantha Narayana

Chairman,

Herbal Products Expert Group (HPEG)

Indian Pharmacopoeia Commission,

Sec 23, Raj Nagar,

Ghaziabad.

Mr. B. Murali,

Sr. Manager - Quality Control

M/s Natural Remedies,

Bangalore

Prof S.S. Handa

Former Director,

Indian Institute of Integrative Medicines

(formerly RRL),

Jammu

Dr. C.K. Katiyar

Chief Executive Officer,

M/s Emami Healthcare,

Kolkata

Dr. Amit Agarwal,

Director- R&D

M/s Natural Remedies

Bangalore

Dr. George Patani,

Director- R&D

M/s Inga Pharmaceuticals Pvt. Ltd.

Mumbai

Mr. Ramakant Harlal Kha,

M/s Nishant Aromatics

424/425, Milan Industrial Estate, Off T J Road,

Cotton Green, Mumbai,

Maharashtra 400033.

Mr. B. Murali

(Affiliation - as above)

Dr. Rahul Singh,

M/s Emami (I) Pvt. Ltd.

687 Anandapur, EM Bypass, Kolkatta 700107

Dr. Hema Lohani

Centre for Aromatic Plants, Selaqui Industrial Area,

Dehradun, Uttarakhand

Ms. Bhuvana Nageswaran

M/s Ultra International Ltd.

64/1, Site 4, Sahibabad Industrial Area,

Ghaziabad, Uttar Pradesh 201010


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IPC Scientific Team

Other Participants

Scientists other than those mentioned above who contributed in the work related to development of

monographs included in IP on Herbs and Herbal Products till today:

Dr. Jai Prakash,

Senior Principal Scientific Officer

Mr Alok Sharma

Scientific Officer

Dr. Manoj Kumar Pandey

Scientific Officer

Dr. M. Kalaivani

Scientific Assistant

Ms. Shruti Rastogi,

Pharmacopoeial Associate

Mr. Hariom Singh,


Mr.Ashish Kumar Kushwaha


Dr. Divya Kaushik


Ms. Neha Singh,


Ms. Amandeep Bhatia,


Ms. B. Gayatri Dr. Anil Thakan

Dr. Anil Kanaujia Dr. G. Trimurtulu

Mr. M. J. Saxena Mr. K. Ravikant

Dr. S.P.S. Khanuja Dr. Beena Bhatt

Mr. Praful Lahorkar Dr. Dharmendra Kushwah

Dr. B. Murali Dr. Vijay Chauhan

Dr. S. Natarajan Dr. Hemant Kumar Sharma

Dr. Abraham Patani Mr. Rajendra M Dobriyal

Dr. George Patani Dr. Ravi Jain

Prof. K. Satyamoorthy Mr. D.K. Ved

Dr. M. Rajani (Late) Dr. Rahul Singh

Dr. Y. K. S. Rathore Mr. Ashish Suthar

Dr. Nancy Pandita Mr. Hariharan

Dr. Padmalatha S. Rai Dr. Ravishankara Bellampalli

Prof. M. N. Nanjan Dr. Deepak

Dr. Ashish Suthar Dr. A.K. Rawat

Dr. Amit Agarwal Dr. Pulok Mukherjee

Dr. C.K.Katiyar Mr. Prakash Itankar

Dr. Ramakant Dr. Ramakant Harlal Kha

Ms. Bhuvana Nageswaran Dr. Hema Lohani

Dr. Vandita Shrivastava


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Acknowledgements

The Indian Pharmacopoeia Commission (IPC) wishes to acknowledge the contributions of all experts,

officials, scientists and academicians involved in bringing out this Herbal Guidance Manual. The

encouragement and support given by Dr. G. N. Singh, Secretary-cum-Scientific Director of the IPC

and DCG (I), is noteworthy. The preliminary draft of this Guidance Manual was prepared by

Dr. D.B.A. Narayana, Chairman, Herbal Products Expert Group and Member, Scientific Body of IPC.

Modifications were done by the IPC scientific team namely Dr. Manoj Kumar Pandey,

Dr. M. Kalaivani, Mr. Hariom Singh, Ms. Shruti Rastogi, Mr Ashish Kushwaha, Dr. Divya Kaushik,

Ms. Neha Singh and Ms. Amandeep Bhatia. Special thanks to Herbal Products Expert Group and the

Chairman who reviewed the draft critically and also to other experts who provided comments on

uploaded draft displayed on the website of IPC www.ipc.gov.in.

The Scientific staff of the IPC provided their valuable scientific inputs including arrangement, fine

editing and formatting of content of this Manual. In particular Dr. D.B.A. Narayana, Dr. Jai Prakash

and Dr. Manoj Kumar Pandey have played a key role in overall review, editing, compilation and

bringing it to present shape.

IPC also places on record the contribution of other participants listed in this Guidance Manual.

Secretarial assistance provided by Ms. Reena Tripathi is thankfully acknowledged. We are thankful to

the printing services officials for their kind support.

The feedback on this Manual will help in further improvement of this document.



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ABBREVIATIONS

BRS : Botanical Reference Substance

CDSCO : Central Drugs Standard Control Organization

CoA : Certificate of Analysis

CV : Coefficient of Variation

FD : Fluorescence Detector

GC : Gas Chromatography

HPEG : Herbal Products Expert Group

HPLC : High Performance Liquid Chromatography

IP : Indian Pharmacopoeia

IPC : Indian Pharmacopoeia Commission

LoD : Loss on Drying

NRA : National Regulatory Authority

PI : Participating Institution

PPM : Parts per million

PRS : Phytochemical Reference Substance

RID : Refractive Index Detector

RS : Reference Substance

SOPs : Standard Operating Procedures


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1. INTRODUCTION

1.1 IP 2014

The seventh edition of Indian Pharmacopoeia (IP 2014) is published by the Indian Pharmacopoeia

Commission (IPC) on behalf of the Government of India, Ministry of Health & Family Welfare. IP

2014 has come into effect from 01.04.2014. As per the provisions of the Drugs & Cosmetics Act 1940

& Rules 1945, IP prescribes the standards for the drugs that are manufactured, imported and stocked,

marketed/distributed in India. The standards of identity, purity and strength prescribed in IP ensure

quality control and quality assurance of the medicines. The standard prescribed in the book applies for

the drugs which have not been included in IP 2014, but included in IP 2010 and its Addendum. The

drugs are to conform the standards prescribed in the IP for the time being in force and its immediately

previous edition.

Herbal medicines have been used by the humanity since time immemorial for various healthcare

needs. Around 80% of the population of developing countries uses herbal medicines for treatment of

various diseases. India is one of the largest producers of medicinal herbs and is called as botanical

garden of the world. With the ever-increasing use of herbal medicines and the global expansion of the

herbal medicines market, quality and safety has become a major concern for the health of the people.

The quality of herbal medicines has a direct impact on their safety and efficacy. Thus, to regulate the

quality standards, various identification and quantification procedures are mentioned in IP 2014.

IP 2014 contains 136 herbal monographs out of which 68 monographs are of raw herbs, 29

monographs of herbal extracts, 39 monographs of processed herbs/ pharmaceutical aids/ formulations.

1.2 IP Addendum 2015

IP Addendum 2015 to IP 2014 is also published by the IPC which has the same authority as IP 2014

and includes the changes and new monographs. It contains 13 new herbal monographs. The effective

date of IP addendum 2015 is 1st April 2015.

1.3 Objective of the Herbal Guidance Manual

This herbal manual is designed to disseminate the practical information and promote the proper use

and development of herbs and herbal products for the benefit of mankind. It is to facilitate the

stakeholders to provide the monographs for inclusion in IP. The details regarding the identification

and testing of the herbs or herbal preparations are provided in the Manual to ensure its quality. This

manual has been developed with a goal of providing assistance to stakeholders i.e. industry, health

care professionals, analysts and researchers on how they can assure high quality procedures for

successful development of the monographs. However, the test methods described in this Herbal


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Guidance Manual are presented as examples of suitable methods for herbal materials. The analysis of

herbal medicines is, however, not restricted to those methods discussed or recommended here. Other

validated techniques may be used in exceptional cases.

Appearance of herbal drug monograph does not mean its approval as a drug under the law; it is to

provide qualitative and quantitative standards of quality for the herbs, its use either as a food item or

as supplement or ingredient, as a drug and/or as an ingredient in cosmetics.

1.4 Scope

Unless otherwise stated, the quality expectations outlined in this document apply to all the herbs and

herbal preparations. This document should be read in conjunction with the IP 2014 and Addendum

2015.

This document can be utilized as a starting point for the development of new monographs by the

various stakeholders for IP. The inclusion and exclusion criteria are mentioned in Chapter 3 of this

Manual.

The guidance provided in this manual will help ensuring quality of the herbal products by the

manufacturers, analysts etc.

1.5 Notes to Readers

The compiled draft of this Guidance Manual for IP was examined by the Herbal Experts Committee

and corrections incorporated as suggested by them. The manual was uploaded on the website of the

Commission www.ipc.gov.in for 45 days for public comments. The comments received from the

stakeholders were examined and then incorporated.

IPC requests all stakeholders to send their feedback on this Guidance Manual. A format for the

feedback is presented at the end of this Manual. Views can be communicated through e-mail also on

the following address:

Email: [email protected]

Website: www.ipc.gov.in

Telephone and fax numbers: +91-120-2783401, 2783392





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2. LIST OF MONOGRAPHS ON HERBALS IN IP 2014 INCLUDING

THOSE IN ADDENDUM 2015

RAW HERBS

(A)

HERBAL EXTRACTS

(B)

PROCESSED HERBS/

PHARMACEUTICAL AIDS/

FORMULATIONS

(C)

1. Acacia

2. Ajwain

3. Amalaki

4. Amaltas

5. Amra

6. Anantmula

7. Arjuna

8. Artemisia

9. Ashwagandha

10. Asthisamhrta

11. Bakuci

12. Bala

13. Bassant

14. Belladona Leaf

15. Bhibhitaki

16. Bhringraj

17. Bhuiamla

18. Birmi

19. Brahmi

20. Coleus

21. Daruharidra Roots

22. Daruharidra Stems

23. Draksha

24. Ergot

25. Garcinia

26. Ginseng

27. Gokhru

28. Gudmar

29. Guduchi

30. Guggul resin

31. Haridra

32. Haritaki

33. Hingu

34. Ispaghula Husk

35. Ivy Leaf

36. Kalmegh

37. Kaunch

38. Kundru

39. Kutki

40. Lasuna

41. Lavang

42. Lodhra

43. Mandukaparni

44. Manjistha

45. Maricha

46. Methi

47. Mirch

48. Nagakesar

49. Neem

50. Pippali Large

51. Pippali Small

52. Punarnava

53. Sahajana Leaf

54. Sahajana Stick

55. Shankhpushpi

56. Sarpagandha

57. Saunf

58. Senna Leaf

59. Senna Pods

60. Shatavari

61. Shati

62. Sunthi

63. Tulasi

64. Valerian Root

65. Vasaka

66. Vidanga

67. Vijayasara

68. Yasti

1. Amla Juice Powder

2. Arjuna Dry Extract

3. Ashwagandha Dry

Extract

4. Bassant Dry Extract

5. Belladonna Dry Extract

6. Belladonna Soft Extract

7. Bhibhitaki Aqueous

extract

8. Brahmi Extract

9. Coleus Dry Extract

10. Garcinia Aqueous

extract

11. Ginseng Dry Extract

12. Gugulipid

13. Haridra Dry extract

14. Haritaki Aqueous extract

15. Haritaki Extract

16. Ivy Leaf Dry Extract

17. Kalmegh Dry Extract

18. Senna Dry Extract

19. Sunthi Extract

20. Tulasi Dry Extract

21. Valerian Dry Extract

22. Vasaka Extarct

23. Yasti Dry Extract

24. Opium

25. Bhuiamla Dry Extract

26. Gudmar Dry Extract

27. Kunduru Dry Extract

28. Mandukaparani Dry

Extract

29. Malt Extract

1. Arachis oil

2. Basil Oil

3. Belladonna Tincture

4. Black pepper oil

5. Caraway Oil

6. Cardamom Oil

7. Castor Oil

8. Clove Bud Oil

9. Clove Leaf Oil

10. Clove Stem Oil

11. Coconut Oil

12. Coriander Oil

13. Cumin Oil

14. Dill Seed Oil

15. Prepared Ergot

16. Eucalyptus oil

17. Guar Gum

18. Hydrogenated Castor Oil

19. Lavender Oil

20. Lemon Grass Oil

21. Lemon Oil

22. Lime Oil

23. Mentha Oil

24. Mentha Arvensis Oil

25. Nutmeg Oil

26. Papain

27. Peppermint Oil

28. Rosemary Oil

29. Sarpagandha Powder

30. Sarpagandha Tablets

31. Shellac

32. Starch

33. Thyme Oil

34. Tolu Balsam

35. Tragacanth

36. Opium powder

37. Ipecac Tincture

38. Gugulipid Tablets

39. Senna Tablets

Gross Total: A+ B+ C =

68 + 29+ 39 =136


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3. INCLUSION/ EXCLUSION CRITERIA OF A HERBAL

MONOGRAPH IN IP Pharmacopoeial herbal monographs contain information on the definition followed by specifications.

The specifications cover the various qualitative and quantitative tests, procedures and acceptance

criteria. The monographs shall employ various validated analytical procedures for the tests that are

feasible to be performed by a trained and experienced analyst without any repetition or development

of new procedure.

3.1 Inclusion Criteria

The following criteria were agreed upon by the Expert Committee:

The herb should be commercially available.

Should be of public interest.

Of clear and defined botany (in case of more than one species/varieties, cultivars of

same species/variety, there could be more than one monograph in IP).

Of known phytochemistry and reported even if not fully.

Some amount of information and /or method for analysis should be available, which

can be used as basis for development and validation. For this knowledge of

“markers”, if available is an advantage.

Sustainable (if the herb is in any regulated list, but knowledge of its sustainability

improvement is ongoing, and is of importance for use, a monograph would be

considered for inclusion).

Knowledge of its safety profiles through known history of its use.

For inclusion of an extract, either the extracts are made using traditional

methods/processes on a commercial scale or standardized extracts that are available

commercially using solvents are selected. For the latter cases, such extracts should

have been in commercial productions and use for at least 15 years, and their safety

profiles are known to the stakeholders.

Herbs should have therapeutic/ prophylactic value.

For finished products offered for sale in market, the products should have been

approved as a drug under Drugs & Cosmetics Act 1940 by CDSCO or by State

Regulatory Authority, as the case may be.

3.2 Exclusion Criteria

Drugs banned in India

Obsolete Drugs


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Drugs considered inappropriate by Indian Pharmacopoeia Commission and

Regulatory Authority.


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4. PROCESS OF IP HERBAL MONOGRAPH DEVELOPMENT AND

CONTENT OF A HERBAL MONOGRAPH

The principles of ‘openness, justice and fairness’ are kept in mind during compiling,

verifying/validating and editing the contents of the monographs in IP.

The herbal drug monographs in IP include crude herbs, processed herbs, herbal materials, herbal

preparations and finished herbal products that contain as active ingredients, the parts of plants, or

plant materials, or combinations thereof. The specific monograph of IP of a herb details the Title and

Synonym if any, Definition, Limits of active ingredient/Marker compounds, Description, Category,

Identification, Chemical Tests, Assay of the marker constituents, Contaminants, Specific Tests,

Storage conditions etc. Monographs are included in IP based on documented quality specifications

and suggestions/feedback from the stakeholders.

The general test methods which are common in nature are cited in the Appendix and are covered in

Volume I of IP. It involves rigorous consultation with experts, verification and validation of data,

identification of monographs and updation on continuous basis as well as responding to the

stakeholder’s queries on the existing monographs.

The flow diagram for herbal monographs development is given below.


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Herbal Monograph Development

Feedback from Stakeholders

Herbal monograph published in IP

Incorporation of valid comments for adoption in Indian Pharmacopoeia

Examination of public comments

Draft monograph displayed on IPC website for the public comments for a period of 45 days

Draft circulated to HPEG expert committee members, editing and formatting of draft monograph is done and forwarded to HPEG review

The participating institution verifies/validates the data and sends the draft monographs to the technical liaison staff of Indian Pharmacopoeia Commission

Sample authenticated and comparison made with reference substance for monograph development

Methods to be used for the Identification and Assay with literature reference are searched for analysis.

Efforts made to obtain 3 samples from different sources

Concurrence of Chairman of HPEG for the items for the monograph development

based on laid down criteria

Screening of proposal / request from stakeholder in the Department of Phytopharmaceuticals for herbal monograph inclusion in Indian Pharmacopoeia


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Content of a Herbal Monograph

The following concepts are important in the development and setting of specifications and should be

provided for each herbal monograph. The monograph should include: Title, Definition, Limits of

active ingredients, Marker compounds, Description, Category, Identification, Chemical Tests, Assay

of the marker constituents, Contaminants, Specific Tests, and Additional Requirements if any.

4.1 Monograph Title

For monographs intended for inclusion in pharmacopoeias, the title of the monograph should include

the Latin binomial nomenclature or Synonym or Common Name whichever is appropriate and is

followed by the name of plant part(s) or plant product (e.g., resin, gum-resin), and where applicable

the processed form.

4.2 Definition

Some or all of the following are usually included in the definition:

the state of the drug: whole, fragmented, peeled, cut, fresh or dried;

the complete scientific name of the plant (genus, species, subspecies, variety, author);

commonly used synonyms may be mentioned

the part or parts of the plant used

where appropriate, the stage in the growth cycle when harvesting takes place, or other

necessary information

wherever possible, the minimum content of quantifiable constituents (either responsible for

the biological activity of the herb (bio-marker) or a chemical compound known to be present

in the herb even if not responsible for biological activity (chemical/ analytical marker)

Herbal drugs very often contain a mixture of related substances, in which case the total

content of quantifiable constituents is determined and expressed as one of the constituents,

usually the major constituent; separate limits may be given for different forms of the drug

(whole/cut)

4.3 Characters

This section contains a brief description of the organoleptic characters of the drug such as colour,

odour, taste etc.

4.4 Category

It includes the therapeutic/prophylactic category of the drug.


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4.5 Identification. The purpose of the Identification in a monograph is to ensure that the article under

examination is in agreement with what is stated in the Definition of the article. All the identifications

mentioned below are not necessarily included: some may be absent when they are not feasible or are

not significant for the purpose of identification. Macroscopic and microscopic requirements of an

herbal monograph should be provided in detail with colour photographs. The detail about

identification is mentioned in Chapter 10.

4.5.1 Macroscopic. The important macroscopic botanical characters of the drug are specified to

permit a clear identification. When two species/subspecies of the same plant are included in

the definition, the individual differences between them are indicated.

4.5.2 Microscopic. It involves gross microscopic examination of the drug and it can be used to

identify the organized/ unorganized drugs by their known histological characters. It is mostly

used for qualitative evaluation of organized crude drugs in entire and powder forms with help

of microscope. It involves using microscope for detecting various cellular tissues and their

arrangements such as trichomes, stomata, starch granules and calcium oxalate crystals etc.

Crude drug can also be identified microscopically by cutting the thin TS (transverse section)/

LS (Longitudinal section) especially in case of wood. Quantitative aspects of microscopy

include study of stomatal number and index, palisade ratio, vein-islet number, size of starch

grains and length of fibers etc.

4.5.3 Fingerprinting. Chromatographic or spectroscopic patterns, sometimes referred to as

“fingerprints”, may be used as standards for identification. These fingerprints can be obtained

by HPLC, UHPLC, capillary electrophoresis, GC, TLC/HPTLC, IR, and Mass Spectroscopy.

The fingerprints must be able to distinguish these materials from other materials with

potential for species substitution and suspected adulteration. The acceptance criteria for

identification tests using chromatographic methods such as HPLC, UHPLC, capillary

electrophoresis or GC methodology must contain a description of the critical features of the

fingerprint chromatograms such as the presence of specified peaks, retention time, their order

of elution, and where possible, their relative abundance. For methods of TLC/HPTLC,

description must include colour and position of the characteristic bands. A colour image of a

typical TLC/HPTLC chromatogram should be provided. A critical aspect of the identification

of herbal materials by separation techniques is the use of reference standards for comparison.

In addition to the Sample solution, a Standard solution containing the reference standard is

chromatographed concomitantly. The reference material used in the Standard solution may be

an Authenticated Botanical Reference Substances (BRS), a reference standard extract, a single

chemical entity, or a standardized mixture of substances.


17

4.6 Tests

4.6.1 Physicochemical Evaluation

It is an important parameter in detecting adulteration or improper handling of drugs. It can serve as a

valuable source of information and provide appropriate standard to establish the quality of herbs.

These are:

Extractable matter :

It is considered useful to determine extractable matter only in herbal drugs where no

constituent suitable for an assay is known or where the material is used to produce a

preparation with a dry residue.

Total ash:

This test is always included unless otherwise justified. It is to be carried out on the powder

drug.

Acid- insoluble ash :

This test may be carried out depending on the nature of the particular herbal drug and is used

to detect unacceptable quantities of certain minerals.

4.6.2 Loss on Drying

Herbal drugs are dried for preservation purposes. If they are insufficiently dried, growth of yeasts or

moulds may occur. It is the loss of weight expressed as percentage w/w resulting from water and

volatile matter of any kind that can be driven off under specified conditions. The limit is specified on

the basis of the results obtained on a reasonable number of varied samples of acceptable quality.

4.6.3 Swelling Index

Applicable to certain hydrocolloid-containing herbal drugs.

4.6.4 Bitterness values

Applicable to herbal drugs containing bitter principles.

4.7 Contaminants- General

4.7.1 Foreign Organic Matter. It is the material consisting of any or all of the following:

Parts of the organs from which the drug is derived other than the parts named in the definition

and description or for the limit are prescribed in the individual monograph.

Any part of organs other than those named in the definition and description.

Matter not coming from the source plant and


18

Moulds, insects or other animal contamination.

Generally a limit of 2% of foreign matter is imposed, unless otherwise prescribed in a specific

monograph. Where a limit for foreign matter greater than 2% is to be prescribed, it is stated in the

specific monograph with an indication of the type of foreign matter. Where necessary, the monograph

should indicate how the foreign matter is identified.

4.7.2 Heavy Metals

The test is prescribed where there is the potential for contamination by heavy metals. The limit of

heavy metals is indicated in the individual monograph in terms of ppm.

4.7.3 Microbial contamination

The Pharmacopoeial monographs should specify the total count of aerobic microorganisms, the total

count of yeasts and molds, and the absence of specific pathogenic bacteria (e.g., Staphylococcus

aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella and Salmonella species)

4.8 Contaminants- Specific

An individual herbal monograph may require certain specifications that are peculiar to that

monograph, especially when safety is an issue. Limits may be set in certain specific monographs for

the characters that are undesirable or have negative botanic characteristics. When one desires a limit

for harmful substances that are present either naturally in the substance or formed as a result of post-

harvest processing practices, such submissions must be accompanied by toxicity data.

4.9 Assay

Wherever possible, an assay is included. Assay is carried out using suitable instruments such as UV-

Visible spectrophotometer, LC, GC or by HPTLC system etc.

4.10 Additional Information

4.10.1 Storage

Storage conditions are applicable unless otherwise specified: store protected from light. Where

applicable, additional specific conditions are given in the individual monograph.

4.10.2 Labelling

Labelling of herbal products includes the label both upon the immediate container and other

associated labeling and written, printed or graphic materials. The label states the Latin binomial

followed by the authorized name; the plant part(s), plant product, or processed form contained in the


19

container or from which the article was derived. Content in percentage of active principles or marker

compounds should be stated. Labelling should be in accordance with the applicable drug laws.


20

5. GENERAL MONOGRAPH FOR HERBS, PROCESSED HERBS AND

HERBAL PRODUCTS

5.1 Herbs and Herbal Products

Herbs and herbal products have been in trade and commerce and are currently used for a variety of

purposes. India has a rich history of use of herbs, processed herbs and formulations containing herbs

both from traditional wisdom as well as cultural usage. Herbs and herbal products are also regulated

by various laws. For the purposes of pharmacopoeial standards various considerations have been

given. This chapter provides a general outline towards the definitions of crude herbs, processed herbs

and herbal formulations.

5.2 Crude Herbs

This term means, unless specified otherwise, mainly whole, fragmented or cut plants, parts of plants,

algae, fungi, and lichen in a form which is not processed. Herbs are usually in dried form, but

sometimes, when specified, may also be in a fresh form. In specific cases exudates which have not

been processed further also are covered under the term herbs. Processing, does not include, normally

expected value addition steps like grading, sizing, removal of weeds or parts of plants other than those

specified herb and removal of adulterants. The term herbs, though botanically generally refer to plants

of specified height and nature, for the purposes of pharmacopoeial reference, shall mean and include

plants and parts of plants not necessarily from herbs and shrubs, but cover the entire range namely

creepers, climbers, trees etc. Each monograph of a herb in the pharmacopoeia shall specify the

scientific name according to binomial system specifying the genus, species, variety and author. In

cases where there are controversial botanical identity, as is seen with mainly herbs known in the

Indian traditional system, the monograph shall specify the official name of the herb along with its

botanical scientific name and guidance is taken from Ayurvedic Pharmacopoeia of India* to decide

the same. In cases where, the same herb is available in different grades or sizes, if found appropriate

and necessary, separate monographs may be introduced in the pharmacopoeia to cover each of them

with appropriate standards. For example -Pippali (large) and Pippali (small).

*Ayurvedic Pharmacopoeia of India, Deptt. of Ayush, Ministry of Health and Family Welfare,

Govt. of India.

In order to improve authentication of botanical identity, especially in those cases where there are

controversial botanical identity or chances of substitution/adulteration is expected, IP has provided

general test method adopting DNA-based identification test. This method has been provided in

appendix 2.2.17 under General Tests. A detailed test method adopting this has been introduced in the


21

monograph of Shatavari to begin with. Testing by this method has been made mandatory only in case

of a dispute.

While deciding to introduce a monograph for a herb in the pharmacopoeia, the criteria that would be

kept in mind, but not limited to are - herbs with specific name and a definitive botanical identity up to

species, availability and usage in trade and commerce, public interest, knowledge of and availability

of a specific chemical compound of well characterized structure [either responsible for the biological

activity of the herb (bio-marker) or a chemical compound known to be present in the herb even if not

responsible for biological activity (chemical/analytical marker)], availability of a quantitative method

for estimation of such a compound, knowledge of safety of the herb, and its sustainability. Herbs

which may figure in a regulated list under appropriate forest and other laws, may still be taken up for

a monograph for inclusion in pharmacopoeia, if there is knowledge of efforts to cultivate or take care

of sustainability issues and /or specific permission is available under law for use of the herb. As

already specified under “General Notices” in the pharmacopoeia, appearance of a monograph does not

mean its approval as a drug under the law. Monograph of a herb in the pharmacopoeia is to provide

qualitative and quantitative standards of quality for the herb for its use either as a food item or food

ingredient or food supplement/ nutraceuticals, as a drug, and/or as an ingredient in cosmetics. Each

such use would need to comply with applicable regulations. Each herb is regarded as one active

substance, irrespective of the knowledge about the active constituents of the herb is available or not.

Herbs may be exposed to low dose gamma radiation for purpose of reducing their microbial

contamination. Herbs treated with low dose gamma radiation shall meet national laws related to such

treatment and shall be labeled as per law.

5.3 Processed Herbs

Processed herbs means preparations obtained by subjecting herbs to treatment such as extraction,

distillation, expression, fractionation, purification, concentration and partial or full fermentation.

Processing may also be done by way of powdering herbs, preparing tincture, preparing extract,

distilling to get essential oils, fatty oils (either expressed or solvent extracted or a blend of both)

expressed juices, extracted exudates, gums and oleo resins, liquid extract where the solvent is

evaporated to yield concentrated semi solid mass or dried mass. Extraction may be performed by

means of appropriate technology such as infusion, maceration, soxhleting, boiling under ambient or

higher pressure, with or without specified enzymes, with or without agitation and combination

thereof. Drying of liquid extracts for removal of the solvent may be done by using various appropriate

technologies like air drying, sun drying, drying under vacuum or with forced air circulation, drying at

low temperature with air circulation, by way of lyophilization or freeze drying. Extracts of herbs may

also be prepared by using carbon dioxide as a solvent-super critical fluid extraction.

Usage of distillates of herbs especially adopting steam distillation is increasing – for use to provide

health benefits/pharmacological actions when used as products for external use as well as in some


22

cases internally used, and also for flavour and fragrance purposes. Such distillates may be referred as

distilled oils. Distilled oils may be used with or without partial fractionation, purification, selective

removal of identified components (De-mentholized Mentha oil etc.) and blending of natural distilled

oil with fractionated oils. Distilled oils are normally packed in tightly closable glass containers or

glass-lined aluminum container or aluminum containers properly labelled and are to be stored and

transported at temperature below 25°. Exposure to heat and light are to be prevented recognizing their

high flammability, and potential oxidation.

Extracts may be liquid extracts and tinctures, semi solid (soft extracts) or solid dry extracts of known

consistency obtained from herbs. Standardized extract, a term commonly employed, would for

pharmacopoeial purposes, mean an extract adjusted within an acceptable tolerance to a given content

of bio-marker or chemical/analytical marker. Standardization may be achieved by adjusting the

extracts with approved inert material or by blending one or more batches of extracts. Wherever

possible, extracts shall specify the defined range of the constituents (bio-marker or chemical/

analytical marker). Extracts not covered in the above description would be defined by the process of

production of the herb to the extract, solvent used and technology applied. The difference between

extracts and tinctures would be, in the type of solvent used for extracting an herb, and tincture would

normally mean an extract where aqueous-ethanol is used as a solvent for extraction. Dry extracts

usually have a loss on drying or water content not greater than 5 per cent w/w, unless specified

otherwise in any monograph. It is normal to extrapolate safety aspects and history of use information

for extracts as long as the process, solvents, extraction ratios are comparable to the processes used in

documented traditional knowledge. Additionally in cases of standardized extracts the inert

excipients(s), if any used for standardization or adjustment of the content of constituents should also

are declared on the label of such extracts. Extracts shall be free from solvent used for extraction and

shall comply with the respective limits as given in Appendix 5.4 (Residual Solvents) of IP Volume I

Harmful and carcinogenic solvents shall not be used for extraction purposes. Solvents and solvent

systems may include use of propylene glycol, glycerin, sorbitol and such other polyhydroxy alcohols,

as long as the content of such polyhydroxy alcohol are within safe limit in the final product.

In cases where extraction and fractionation process leads to preparation of an extract, which consists

of a single chemical compound of more than at least 70 per cent purity, such extracts shall be treated

as an active pharmaceutical ingredient or a food additive or a cosmetic ingredient and would be

required to meet relevant laws.

Extracts may also be offered as purified or enriched extracts. Such extract of a herb is processed in

such a way to provide higher than normal proportion of the active constituent (s) of the herb as long as

the active constituent (s) is/are known. Such purified or enriched extracts may contain additional

valuable components which may provide specific properties like enhanced efficacy or stability or

solubility and availability of the active constituent (s). Purified and enriched extracts may also be


23

prepared to reduce or remove other specific compound or group of compounds that is scientifically

considered undesirable in the herb extracts. Pharmacological, toxicological, pharmaceutical

considerations need to be applied while preparing such purified or enriched extracts. Mixed extracts

may also be offered which would cover combination of more than one herb extract for purposes of

providing simplification or economical way to manufacture herbal formulations.

Herbs may also be extracted using vegetable oils (approved by Food Law) for extraction purposes and

such extracts shall specify the oil used for processing.

Approved preservatives or preservatives system may be used during preparation of extracts. The

names of such preservatives used which would remain in the final extract shall be listed on the label

of such extract, and the proportion of preservatives used shall not exceed normally accepted safe

limits of their usage as per relevant laws or pharmacopoeial standards. No artificial colours may be

used in extracts of herbs unless and otherwise specified in the specific monograph. Only approved

colours shall be used.

Extracts may be exposed to ethylene oxide fumigation or low dose gamma radiation for purposes of

reducing their microbial contamination. In cases where they are fumigated, the final extracts exposed

shall meet residual levels of ethylene oxide limits as applicable. Herbs treated with low dose gamma

radiation shall meet national laws related to such treatment and shall be labelled as per law.

5.4 Herbal Formulations

Herbal formulation shall mean a dosage form consisting of one or more herbs or processed herb(s) in

specified quantities to provide specific nutritional, cosmetic benefits, and/or other benefits meant for

use to diagnose treat, mitigate diseases of human beings or animals and/or to alter the structure or

physiology of human beings or animals. Dosage forms commonly employed for food or cosmetic or

pharmaceuticals may be employed to formulate one or more herb or processed herbs. Dosage forms

known in traditional medicines may also be adopted for preparing herbal formulations, either for

external use or for internal administration. Adequate consideration for uniform distribution of herb or

processed herbs as well as stability of the same in the dosage form shall be provided during

formulation development.

Herbal formulation shall be labelled to comply with relevant labelling requirements under food or

drug or cosmetics laws as applicable. Additionally, adequate information shall be provided on label of

such formulations to include the name of the herb, parts used, nature and type of extract or processed

herb used, extraction ratios, quantity per unit dose or per serving, name (s) of inert excipients used

and any preservatives added shall be provided on the label.

Appearance of a monograph of a herbal formulation in the pharmacopoeia does not mean its approval

as a drug under the law. Monograph of a herbal formulation in the pharmacopoeia is to provide


24

qualitative and quantitative standards of quality for the formulation for its use either as a food item or

food ingredient or food supplement/ nutraceuticals, as a drug and / or as a cosmetic. Each such use

would need to comply with applicable regulations. Each extract is regarded as one active substance

irrespective of the knowledge about the active constituents of the herb is available or not.


25

6. STANDARD OPERATING PROCEDURE FOR PREPARATION OF

MONOGRAPHS OF HERBS, PROCESSED HERBS AND HERBAL

PRODUCTS IN IP

Standard Operating Procedure (SOP) of herbs, processed herbs and herbal products monographs for

submission to IPC is described below:

6.1 Requirements for inclusion of photograph of an herb /part of the herb

a) An herbal monograph in IP provides photograph of the herb. Such a photograph shall provide a

clear visual depiction of the herb, part of the herb.

b) A photograph of the herb shall appear immediately after the title/synonym in the monograph.

c) An authentic sample of herb/part of a herb, properly cleaned, kept within a grid printed on a

paper which gives it the size denotation as illustrated in Table 1 shall be photographed using an

appropriate camera, with a minimum of 3 megapixels capacity. The pieces should be clearly

visible.

d) Alternatively, place such a sample on a glass plate which can be illuminated from below using

a suitable lamp and photograph it from a suitable distance from the top with proper focus.

While doing so depending on the colour of the backgrounds like butter paper, white paper,

black paper etc. may be used suitably.

e) The photograph shall be saved and reproduced in the IP as a composite photograph occupying a

size of 8 x 6 cm.

f) Alternatively, the same may also be reproduced in such a way to cover the requisite units

occupying 5 x 6 cm and a photograph of 1 or maximum 2 single units in a “close up” mode

occupying 3 x 6 cm size. In no case any photograph shall exceed 8 x 6 cm size. Table 1.

Describe the number of units of each material to be taken for the photograph.

While taking the photograph, name of the botanical as given in the title of the monograph (this shall

be printed in “Times New Roman” Font, with a size of 14) shall be kept along with the units of

botanicals


26

Table 1: Description of number of units of each herbal material

Category No. of

Units

Category No. of

Units

Woody and available in large pieces –

stem, wood, and heartwood, woody roots

(Eg. Deodar, Erandmool)

4-6 Stems and roots with smaller

diameters

(Eg. Ephedra, Manjistha, Kutaki)

8-14

Leafy and creepers cut into parts

(Eg. Bhringraj, Neem)

10 to 12 Stigma, Style, Anthers, Small Petals,

Buds

(Eg.Keshar, Lavang)

20-40

Fleshy Dried Rhizomes

(Eg.Vidarikand, Varahi)

4-8 Minute seeds and parts of seeds

(Eg.Vakuchi, Isabgol)

More than

40

Flowers, Larger Petals, Small Fruits

(Eg.Japakusum, Kusumphool)

10-20 Resins, Gums in dried form

(Eg. Heeng, Babool)

4-8

Bark cut into pieces

(Eg. Arjuna, Kutaz)

3-8 Minute parts like epidermal hair

(Eg. Kamela)

5-10

Example: Photograph of Arjuna


27

Grid to be used to place the herbs for photograph (Each block is of 1 cm2)

Empty Grid

6.2 SOP for TLC/HPTLC profile

a) A “Typical TLC/HPTLC profile” depicts the results of the test for identification/assay used.

A photograph of such TLC profile should be submitted on a separate sheet.

b) Identification tests by TLC/HPTLC shall be performed as per specification given in the

respective monograph

c) As a common practice, the plate shall be of at least 5 cm width and 10 cm height. In this

dimension 2 bands each of 10 mm width would be spotted. As a rule the extreme left track

(track 1) shall always be a Botanical Reference Substance (BRS)/ Phytochemical Reference

Substance (PRS). The track 2 of 10 mm width band shall be of a solution of material under

examination. All the bands shall be applied at a height of 20 mm from the base of the plate.

During development, the solvent front shall be allowed to move to at 80% of the plate height.

d) A photo-documentation of the plate developed as above, after visualization under UV 254 nm

and 366 nm, and/or by any derivitizing or by spraying reagents shall be photographed.

e) On top of all pages where such typical TLC/HPTLC profiles are reproduced following text

should be written.

“Reproduce below is Typical TLC Profiles of herbs for which a monograph appear in IP. Each

typical profile bears the name of the monograph below the profile.”


28

Example: Pippali (Small)

Reproduce below is Typical TLC Profiles of herbs for which a monograph appear in IP. Each typical

profile bears the name of the monograph below the profile.

(Under UV light at 254 nm) (Under UV light at 366 nm)

6.3 SOP for A typical “HPLC / GC chromatogram”

a) A HPLC/GC Chromatograms should be submitted on a separate sheet.

b) Separate chromatograms should be submitted for respective PRS and sample under

examination. The peak of the PRS/ compound under examination shall be labelled

accordingly in the respective chromatograms, suffixed by a small arrow pointing to the peak.

Other peaks that may appear in the chromatogram, whose chemical identity is not known,

need not be labeled.

c) While supplying such HPLC/GC chromatograms please ensure that the chromatogram

should;

Contain appropriate scale in X and Y axis with respective units.

Not contain any notations given by the equipments like date, sample details, annotation

and all such other matter.

Not contain names of analyst, firms that may appear as a routine part due to settings. On

top of all pages where such typical chromatograms are reproduced, following text should

be written:

“Reproduce below is Typical chromatograms of herbs for which a monograph appear in IP. Each

typical chromatogram bears the name of the monograph below it. Methods adopted and the

interpretations of the chromatogram are detailed in the monograph.”

(Under day light after spraying with

vanillin sulphuric acid reagent)


29

Example: Typical HPLC Chromatogram of Shankhpushpi

Reproduce below is Typical chromatograms of herbs for which a monograph appear in IP. Each

typical chromatogram bears the name of the monograph below it. Methods adopted and the

interpretations of the chromatogram are detailed in the monograph

HPLC chromatogram of PRS

HPLC chromatogram of Shankhpushpi


30

Example: Typical GC chromatogram of Nigella sativa”

“Reproduced below are Typical chromatograms of herbs for which a monograph appear in this

edition of IP”.

GC chromatogram of Nigella sativa

p-C

ym

ene

Res

pon

se (

mV

)

α-

Pin

ene


31

7. STANDARD OPERATING PROCEDURE FOR HERBAL EXTRACTS

MONOGRAPHS

Requirements for Developing Monographs for Inclusion in IP

7.1 Purpose

This document provides guidance for preparation of monographs for herbal extracts (one of the types

of processed herbs) for inclusion in the Indian Pharmacopoeia”.

7.2 Definition

a) Processed herbs have been explained in detail in the General Requirements for “Herbs and

Herbal Products” in IP 2014, Vol 3; page 3169. “Extracts” may refer to either an extract of a

herb prepared by using water or organic solvent as a solvent for extraction.

b) As per IP 2014’s General Requirements, in the extract of the herb prepared using water as a

solvent for extraction, it is appropriate to extrapolate the history of safe use, if the herb is also

known in Ayurveda or other traditional systems of medicines.

c) For purposes of IP, extract of the herb prepared using an organic solvent for extraction is

referred as “Solvent extract”. As per IP 2014’s General Requirements, for such extracts, it

may not be appropriate to extrapolate the history of safe use from Ayurveda or other

traditional systems of medicines, as the processing methods /usage methods differ from that

known in tradition.

For such extracts the following additional requirements need to be met for inclusion in IP.

The solvents used should be safe, avoid chloroform and such solvents. The final product

(extracts) needs to conform to residual solvent levels requirements of the pharmacopoeia.

The extracts being included in IP, should have been in commercial usage for at least a

period of 10 years. The stakeholder who shares the data and provides information to help

IP include the monograph need to certify that the particular extract/process has been

followed by them. Additionally the stakeholders need to inform IP committee regarding

any safety/toxicology data.

In addition to steps of solvent extraction, if the processing of the herb involves other

steps like salting out, (as sodium/potassium/calcium salts) or other esterification, such

extracts may also be considered for inclusion in IP, subject to them meeting requirements

under section above.

Stakeholders should not suggest an extract whose process has undergone changes and

have not been in market for at least 10 years. This ten years commercial usage

requirement means that the particular candidate extract has been manufactured on a


32

commercial scale and has been supplied to industry in India, or abroad for manufacture

of any category of products for human or animal consumption.

The decision of inclusion of any monograph for any extract monograph however is the

prerogative of Scientific Body of IPC. The stakeholders would need to make available

the safety information available with them for inspection to herbal of IPC. They need not

provide copies to herbal expert group as it would be the property of the firm. However,

the herbal expert group would need to see the data before agreeing for inclusion or

otherwise of the proposed extracts monograph. IPC’s herbal expert group may also

request for any additional information to substantiate the long years of usage of the

extract under consideration.

It may also be appreciated that the mere inclusion of a monograph for any extracts in IP

does not necessarily mean its approval as a drug. (Please refer to General Monograph on

Herbs, processed herbs and herbal products, IP 2014, Vol 3, pg. 3170).

The process of developing a monograph shall be the same as adopted by IP for raw herbs or for

finished herbal products. For this purposes samples to be tested should be from three commercial

batches and not from small batches made in the laboratory. Batches made in pilot plants can be used

provided the batch sizes are at least 15% of the normal commercial batch sizes intended or adopted

for commercial purposes. Samples of three such batches shall be involved in development of draft

monographs and theses samples will also undergo “RING TESTING”, with two more labs including

Indian Pharmacopoeia Laboratory. The final monograph, test methods and the tolerance limits, shall

be decided based on application of scientific thinking to the results of ring testing.

For each monograph on extracts there shall be a mandatory testing for quantitative assay of at least

one or more marker(s) compounds which are well characterized chemical substance(s).

At this stage testing for at least one marker is to be relied upon, and in cases where scientific

knowledge exists /or is developed for more than one marker that can be tested, such multiple marker

testing may be adopted and prescribed by IP:

a) The monograph of extract shall at the beginning prescribe the quantitative contents (minimum

or limits) for such marker(s).

b) These marker(s) may be either those that are linked to biological activity or just analytical

marker(s).

c) The marker(s) used for testing need not be the same, and can be different basis the presence of

the compounds and the scientific knowledge.

d) Test(s) for absence of any marker may also be considered & included, if felt necessary to

control quality or safety through such testing.


33

e) Other physicochemical tests that can give information for objective assessment of quality of

the extract would be included in the Standard tests to be performed.

f) Other requirements like microbial contamination and heavy metals as per IP shall also be

applicable.

g) The monographs shall also mention and include any tests that may be required to check for

the excipients added, if any, if there is a need felt.

The final monograph developer shall also provide to IP, the chromatograms for inclusion in IP under

the relevant section as being done for herbs, as guide to users.

Example of Extract Monograph in IP 2014

Arjuna Dry Extract

Arjuna Dry Extract is obtained by extracting Arjuna

(Terminalia arjuna Wight and Arrn,

Fam. Combretaceae) bark with methanol or any other

suitable solvent and evaporation of solvent.

Arjuna Dry Extract contains not less than 90.0 per

cent w/w and not more than 110.0 per cent w/w of

stated amount of the arjunolic acid on the dried basis.

It may contain suitable added substances.

Category. Antihyperlipidaemic, antihypertensive,

astringent, cardioprotective, hrdroga.

Usual strength. 60 per cent w/w.

Description. A light brown to beige powder with or

without red tinge.

Identification

A. Determine by thin-layer chromatography (2.4.17),

coating the plate with silica gel GF254.

Mobile phase. A mixture of 92 volumes of chloroform

and 8 volumes of methanol.

Test solution. Dissolve 50 mg of the extract under

examination with 50.0 ml of methanol and filter.

Reference solution. A 0.1 per cent w/w solution of

arjunolic acid RS in the methanol.

Apply to the plate 5 µl of each solution as bands 10

mm by 2 mm. Allow the mobile phase to rise 8 cm.

Dry the plate in air and spray with anisaldehyde

sulphuric acid reagent. Heat the plate at 110° for 10

minutes and examine the plate under ultraviolet light

at 365 nm and day light. The chromatographic profile

of the test solution is similar to that of the reference

solution.

B. Determine by thin-layer chromatography (2.4.17),


Mobile phase. A mixture of 35 volumes of

chloroform, 10 volumes of methanol and 2 volumes of

water.




arjugenin RS in the methanol.



Dry the plate in air and spray with vanilline sulphuric

acid reagent. Heat the plate at 110° for 10 minutes


34

and examine the plate under ultraviolet light at 365

nm and day light. The chromatographic profile of the

test solution is similar to that of the reference solution.

Tests

Ethanol-soluble extractive (2.6.2). Not less than

80.0 per cent.

Total ash (2.3.19). Not more than 5.0 per cent.

Heavy metals (2.3.13). 1.0 g complies with the limit

test for heavy metals, Method B (20 ppm).

Loss on drying (2.4.19). Not more than 5.0 per cent,

determined on 1 g by drying in an oven at 105°.

Microbial contamination (2.2.9). Complies with the

microbial contamination tests.

Assay. Determine by liquid chromatography (2.4.14).

Test solution. Shake a quantity of the extract under

examination containing about 50 mg of arjunolic acid

in 50.0 ml of the methanol, filter.

Reference solution. A 0.1 per cent w/v solution of

arjunolic acid RS in methanol.

Chromatographic system

– a stainless steel column 25 cm x 4.6 mm

packed with octadecylsilane bonded to porous

silica (5 µm),

– mobile phase: a mixture of 35 volumes of 5 mM

α- cyclodextrin and 65 volumes of methanol,

– flow rate: 1 ml per minute,

– spectrophotometer set at 205 nm,

– injection volume: 20 µl.

Inject the reference solution. The test is not valid

unless the relative standard deviation for replicate

injections is not more than 2.0 per cent.

Inject the reference solution and the test solution.

Calculate the content of the arjunolic acid in the

extract.

Storage. Store protected from heat and moisture.


35

8. STANDARD OPERATING PROCEDURE FOR PREPARATION,

QUALIFICATION, CERTIFICATION & SUPPLY OF BOTANICAL

REFERENCE SUBSTANCES (BRS)

8.1 Purpose

This SOP provides the procedure for preparation, qualification and supply of “Botanical Reference

Substance” (hereinafter referred to as BRS) to be used for identification of commercial supplies of

raw material, of botanical origin, as per the procedures mentioned in Indian Pharmacopoeia.

8.2 Definition

Botanical Reference Substance is a standard whose botanical identity and genuineness has been well

established to both genus and species level.

8.3 Need

It is to be used as a reference material for comparison and confirming the identity of the commercial

supplies of the respective botanical as prescribed under tests for identity in the monograph.

Compliance to identity test using microscopic, chromatographic (TLC / HPTLC fingerprint) and other

specified tests will involve use of BRS. The BRS generally has a shelf life of 2 years unless otherwise

stated. Each BRS should be supplied with documentation describing the above characteristic features.

8.4 Scope

Applicable to all the laboratories who are involved in generating ‘BRS’, who will prepare them for

Indian Pharmacopoeia Commission (IPC), who will certify the same prior to issue.

8.5 Responsibility

IPC: To appoint and authorize one or more laboratories to prepare specific (one or more)

BRS.

Appointed Laboratory: To prepare specific (one or more) BRS as per the procedure described

below and supply the same to IPC from time to time.

IPC to certify the quality of BRS and make them available to the users along with COA

(Certificate of analysis).

IPC to periodically upgrade or revalidate the available BRS.

IPC to make public, the availability of a list of all BRS and their current lot along with price.

8.6 Procedure for appointed Laboratory

a) Collect about 4.0 Kg of raw material after botanical authentication. Botanical authentication will

be done to confirm the genus and species of the botanical raw material. Botanical authentication is

to be done through a qualified and experienced Botanist/Pharmacognosist of any recognized


36

Institutions who would issue a certificate of verification on his Institution’s letter head. Such

authentication should be done at least from two crude drug verification laboratories like

CSIR-National Institute of Science Communication and Information Resources, Dr. KS

Krishnan Marg, Pusa IARI Campus, New Delhi-110 012

CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow – 226001, UP

Agharkar Research Institute, Gopal Ganesh Agharkar Road, Pune – 411004

Jawaharlal Nehru Ayurvedic Medicinal Plants Garden, Near Gandhi Bhavan,

Kothrud, Pune- 411 029, Maharashtra.

Bombay Natural History Society, Hornbill House, Dr. Sálim Ali Chowk, Shaheed Bhagat

Singh Road, Mumbai - 400 023.

b) Prepare at least two specimen of the said plant material out of the 2.0 kg raw material

obtained, label them properly and preserve for future reference.

c) Dry the material in an appropriate manner to approximately 5% w/w moisture content.

Document the passport data including processing details. In case the dried material is

procured from a supplier, the passport data will not involve any processing details.

d) Perform the test(s) for identity as prescribed in the monograph of particular botanical in

Indian Pharmacopoeia. Material which meets the requirement(s) will only be taken up for

processing further. Document all the tests performed, their results and the inference properly

and generate the certificate of analysis (COA) duly signed by the analyst(s) who performed

the analysis and authenticated by the Head of the Laboratory.

e) Powder the raw material to fine size (to pass through 22#sieve) using previously cleaned and

dried suitable grinder free from contamination.

f) Distribute approximately 2 g depending upon the bulk density of the material, into either

manually or using suitable filling equipment such as in a cleaned, dried vial free from any

contamination. For this purpose amber colored USP type 1 Glass vials generally used for

injectables may be used. Vials of size from 2, 5, 10 and 30 ml as required may be used to

hold 2 g of the material without leaving too much of air head space. The mouth of the vial

should be plugged with the suitable size rubber plug previously cleaned and the mouth sealed

with aluminum “Tear Down Seals”. Tear down Seal can be easily applied and sealed on the

mouth of the vials either with a hand sealer or with mechanical sealing machine. Each such

vial containing the powdered material shall be affixed with a label containing the following

information.


37

BOTANICAL REFERENCE SUBSTANCE

Name of BRS, IP (as given in IP Monograph):

Lot No: BM IL DDMMYY Date of Packing: June 15

Storage: Cool and dry place

========================================

Indian Pharmacopoeia Commission, Ghaziabad

Self-adhesive sticker label may be used on which information given below printed using a PC

or otherwise.

g) The Lot No should consist of 4 letters and 6 digits. The first two letters representing the Name

of the botanical, next two letters representing the name of the laboratory, followed by 2 digits

of date, 2 digits representing the month and last 2 digits representing the year of packaging

the BRS.

Eg. Lot No. “BM IL DDMMYY represents “Bacopa monniera, Indian Laboratory” and

the BRS has been packed on the respective date in format DDMMYY, where DD

represents Date, MM represents Month and YY represents Year.

h) Pack the sealed vials in suitable shipper with honeycomb partition as per the requirement of

size and shape of the radiation lab. Most of the existing radiation agencies accept 60 x 43 x 34

cm cartons. Affix copies of above label, in suitable size, at least on two panels of each

Shipper.

i) Send the entire shipper properly sealed and labeled as above to a center for gamma radiation

at 6- 14 k Gy along with suitable dispatch documents.

j) Collect the shippers after irradiation along with certificate of irradiation from the irradiation

centers.

k) Collect 10 irradiated BRS samples and preserve them as Control sample. Dispatch the rest of

the irradiated samples to IPC along with following documents.

Botanical Authentication certificates

Certificate of analysis

Certificate of irradiation issued by irradiating agency

Packing Chalan and in-voice

Certificate for transportation (certifying that material is non-hazardous etc)

Labelling requirement for candidate Botanical Reference Substance


38

7.7 Procedure for IPC

a) On receipt of the consignment cross check the material received against

documents/certificates received.

b) Take adequate samples randomly from the shippers and perform identity test(s) in duplicate

as per standards given in Indian Pharmacopoeia.

c) Upon the samples meeting the requirements of IP, the consignment is ready for certification

as BRS. The COA is prepared as discussed earlier.

d) Upon conforming the materials as BRS, each vial is to be affixed a prior printed “SEAL”

which reads as below-

e) “CERTIFIED BRS – IPC”, such seals need to be affixed in such a way that part of seal

overlaps the label affixed by the appointed laboratory. A suggestive specimen of such seal is

given below. A hologram is also to be added to make sure duplication does not take place.

BOTANICAL REFERENCE SUBSTANCE

Name of BRS, IP (as given in IP Monograph):

Lot No: BOOL/210706 Date of Packing: July 06


=======================================


f) IPC will store the certified BRS samples consignment between “Temp 2-15 0C and 60% RH”.

g) IPC shall supply the requisite no of BRS packs as per order along with a copy of the COA.

7.8 Relevant documents to be maintained by the Appointed Laboratory.

Two reference samples of initial crude botanical.

Authentication certificates

Passport data

Irradiation certificate

COA

IPC


39

9. STANDARD OPERATING PROCEDURE FOR PREPARATION,

QUALIFICATION, CERTIFICATION AND SUPPLY OF

PHYTOCHEMICAL REFERENCE SUBSTANCES (PRS)

9.1 Procedure for appointed Laboratory:

9.1.1 Perform a literature survey, identify and shortlist one or more relevant publications

which can form the basis for a procedure to be adopted to isolate the unique chemical

compound with known structure identified to be used as a PRS. Write down an

approach note/ block diagram of the steps involved in isolating the compound. If

available, note down the percentage of the compound reported in the literature from

that particular plant material.

9.1.2 Collect requisite quantity of fresh raw material (adequate to provide about 1000 mg

of PRS) after botanical authentication. Botanical authentication will be done to

confirm the genus and species of the botanical raw material. Botanical authentication

is to be done through a qualified and experienced Botanist/Pharmacognosist of any

recognized Institutions who would issue a certificate of verification on his

Institution’s letter head. Such authentication should be done at least from two crude

drug verification laboratories like –

CSIR-National Institute of Science Communication and Information Resources, Dr. KS

Krishnan Marg, Pusa IARI Campus, New Delhi-110 012

Agharkar Research Institute, Gopal Ganesh Agharkar Road, Pune – 411004

Jawaharlal Nehru Ayurvedic Medicinal Plants Garden, Near Gandhi Bhavan, Kothrud,

Pune-411 029, Maharashtra.

9.1.3 Process the raw material adopting the procedure as mentioned. Use solvents/solvents

mixtures and relevant chromatographic techniques which may involve column

chromatography or flash chromatography or semi-prep HPLC or GC. Collect the

relevant fractions which are expected to contain the compound to be isolated and

process them to obtain a mixture which is predominantly rich in the desired PRS.

Perform TLC and/or any other test at different stages of such separation to guide that

the process is moving in the right direction.

9.1.4 Purify the above fraction containing the desired PRS using column chromatography

/crystallization technique /preparative HPLC depending on the quantity of the

compound present in the fraction.

9.1.5 Assess the purity of the isolated compound using different chromatographic

techniques like TLC / HPLC / GC etc. If the purity of the compound is less than 98%

w/w, repeat the process as in 9.1.3 including any simple re-crystallization steps


40

involving the relevant solvents of analytical reagents grade. Assess the purity again

to confirm greater than 98% purity before proceeding to the next stage.

9.1.6 Determine the structure of the compound adopting all necessary spectroscopic and

other measurements techniques including, but not limiting to CHN analysis, MS, IR,

UV, NMR, Specific rotation, chiral testing etc.

9.1.7 Prepare a report (COA) documenting the purity testing as well as all the relevant

structure determination testing along with the results obtained, interpretation and

attaching copies of purity and structure determination data.

9.1.8 In those cases, where the PRS compound is being prepared by a synthetic process

(and not isolated from the plant material as above), document the steps involved in

the synthetic process, purification of the final compound obtained as well as its purity

and structure confirmation.

9.1.9 Distribute either manually or using suitable filling equipment (previously cleaned and

dried, free from any contamination) approximately 10 mg (or a quantity as specified

by IPC) into previously cleaned and dried glass vials of suitable size depending upon

the bulk density of the material. Flush the vials with slow stream of nitrogen to

replace the air in the Vials and close with rubber bungs. For this purpose amber

colored USP type 1 Glass vials generally used for injectables may be used. Vials

from size of 2, 5, 10 ml as required may be used to hold the material without leaving

too much of air head space. The mouth of the vial should be plugged with the suitable

size previously cleaned rubber plug and the mouth sealed with aluminum “Tear

Down Seals”. Tear Down Seal can be easily applied and sealed on the mouth of the

vials either with a hand sealer or with mechanical sealing machine. Each such

container containing the PRS shall be affixed with a label containing the following

information.

Self-adhesive sticker label may be used on which information given above printed using a PC or

otherwise.

Labelling requirement for candidate Phytochemical Reference Substance

PHYTOCHEMICAL REFERENCE SUBSTANCE ___mg

Name of PRS, IP (as given in IP Monograph):

Lot No: BM IL DDMMYY Date of Packing: June 15


========================================



41

9.1.10 The Lot no should consist of 4 letters and 6 digits. The first two letters representing

the Name of the botanical, next two letters representing the name of the laboratory,

followed by 2 digits of date, 2 digits representing the month and last 2 digits

representing the year of packaging the PRS.

Eg. Lot no. “BM IL DDMMYY represents “Bacopa monniera, Indian Laboratory” and

the PRS has been packed on the respective date in format DDMMYY, where DD

represents Date, MM represents Month and YY represents Year.

9.1.11 Pack the sealed vials in suitable shipper with honeycomb partition as per the

requirement. Affix copies of above label, in suitable size, at least on two panels of

each Shipper.

9.1.12 Preserve 5 PRS samples as Control sample. Dispatch the rest of the PRS vials to IPC

along with following documents.

Certificate of analysis – Original (Purity and structure confirmation test results along

with copies of all confirmatory test records like spectra and chromatograms)

Packing Chalan and in-voice (original two copies)

Certificate for transportation (certifying that material is non-hazardous etc)

9.2 Procedure for IPC

9.2.1 On receipt of the consignment cross check the material received against

documents/certificates received.

9.2.2 Take adequate samples randomly from the shippers and perform pre-decided test(s) so

as to confirm the purity/structure of the PRS tubes supplied by the appointed

laboratories in duplicate.

9.2.3 Upon the samples meeting the requirements or purity and identity, the consignment is

ready for certification as PRS. Document all the tests performed, their results and the

inference properly and generate the certificate of analysis (COA) duly signed by the

analyst(s) who performed the analysis and authenticated by the Head of the

Laboratory.

9.2.4 Upon conforming the materials as PRS, each tube is to be affixed a prior printed

“SEAL” which reads as below-


42

“CERTIFIED PRS – IPC”, such seals need to be affixed in such a way that part of seal

overlaps the label affixed by the appointed laboratory. A suggestive specimen of such seal is

given below. A hologram is also to be added to make sure duplication does not take place.

9.2.5 IPC will store the certified PRS samples consignment between “Temp 2-15oC and

60% RH” OR at Room Temperature if the PRS would be stable at RT.

9.2.6

9.2.7 IPC shall supply the requisite no of PRS packs as per order along with a copy of the

COA.

9.3 Relevant documents to be maintained by the Appointed Laboratory

a) Spectra and Chromatograms containing data regarding purity and structural conformation

b) COA

c) Relevant literature data and the return down procedure for isolation and purification and/or

the procedure for preparing the PRS by a synthetic process respectively.

PHYTOCHEMICAL REFERENCE SUBSTANCE

Name of PRS, IP (as given in IP Monograph):

Lot No: BM IP 04062015 Date of Packing: June 15


========================================


IPC


43

10. IDENTIFICATION OF HERBAL MATERIALS

The identification of herbal materials is done with respect to macroscopic, microscopic characteristics

and chromatographic procedures etc. An examination to determine these characteristics are the first

step towards establishing the identity and purity of such materials, and should be carried out prior to

any other tests. Wherever possible, authentic specimens of the materials and samples should be

available to serve as a reference.

These tests not only serve to ensure the accurate identification of the herbal material, but must be able

to distinguish it from related species that may pose potential for species substitution or adulteration.

For development of a monograph it is necessary to perform a comparative analysis between the herbal

material and the literature reference. The morphological testing must be done by a person suitably

qualified and experienced.

Macroscopic identity of herbal materials is based on shape, size, colour, surface characteristics,

texture, fracture characteristics and appearance of the cut surface.


44

IDENTIFICATION OF HERBAL MATERIAL

Monograph available in other Pharmacopoeias

Yes No

Compliance with all

identification tests (macroscopic,

microscopic and

chromatographic method) in

available pharmacopoeial

references

ant Pharmacopoeia

Options available

Comparison against a

literature source (articles

in journals, in-house

generated data)

Comparison against an

Authenticated reference

Material

Analyst must perform a

comparative analysis

between the herbal

material and literature

reference

The comparison must

include three or more

of the following:

Macroscopical

characters

Microscopical

characters

Chromatographic

procedures

Chemical tests

Analyst must perform a

comparative analysis

between the herbal

material and literature

reference


45

10.1 MACROSCOPIC EVALUATION

Macroscopic evaluation on the basis of following parameters.

Size

A graduated ruler in millimeters is adequate for the measurement of the length, width and thickness

of crude materials. Small seeds and fruits may be measured by aligning 10 of them on a sheet of

calibrated paper, with 1 mm spacing between lines, and dividing the result by 10.

Colour

Visually examine the untreated sample under diffuse daylight. If necessary, an artificial light source

with wavelengths similar to those of daylight may be used. The colored the sample should be

compared with that of a reference sample.

Surface characteristics, texture and fracture characteristics

Examine the untreated sample. If necessary, a magnifying lens (6x to 10x) may be used. Wetting with

water or reagents, as required, may be necessary to observe the characteristics of a cut surface. Touch

the material to determine if it is soft or hard; bend and rupture it to obtain information on brittleness

and the appearance of the fracture plane — whether it is fibrous, smooth, rough, granular, etc.

Based upon the anatomical structure of the plant, the parts having therapeutic action commonly used

are leaves, barks, flowers, fruits, seeds, wood, aerial parts etc. The characteristics of the individual

group are described below:

10.1.1 Aerial Parts: Aerial parts consist of stems and leaves and sometimes associated with flowers

and fruits. These materials can be detected based on the following description:

A) Description of Aerial Stem. The size, shape, colour, texture - smooth or rigid, hair present or

not etc.

B) Position and arrangement of Leaves.

Radical Arising from the crown of the root

Cauline Arising from the aerial stem

Adnation Fusion of part of leaf with stem

Alternate Leaves arise from stem in alternate manner e.g. Lobelia

species

Opposite Leaves arise in pairs alternately at right angles to the


46

stem.

10.1.2 Leaves or Leaflets

A) Leaf base: Stipulate or estipulate

B) Surface appearance and texture:

Glabrous Surface is free from hair

Pubescent Surface is hairy

Glandular Where long and distinct hairs or glandular hairs are present

Punctuate When surface is dotted with oil glands

The texture can be brittle, cariaceous, papery, fleshy etc.

C) Lamina Structure:

Lamina could be described in terms of margin, incision, composition, apex, base, and venation.

10.1.3 Inflorescence and Flowers

A) Type of Inflorescence. Racemose, cymose, mixed or any other type.

B) Axis or Receptacle of Inflorescence. The main axis of inflorescence is called the rachis while

branches bearing flower clusters and individual flowers are termed peduncles and pedicles

respectively.

C) Type of flower.

1. Monocotyledon or dicotyledon.

2. Unisexual or hermaphrodite.

3. Regular or zygomorphic.

4. Hypogynous, perigynous or epigynous.

D) Calyx and Corolla: It may be polysepalous or gamosepalous; persistent or caduceus; the

description about colour, shape, absence or presence of hair etc.

10.1.4 Subterranean Organs

Crude drugs from subterranean organs include stem structures such as corms, bulbs, stem-tubers,

rhizomes and root structures such as root tubers or adventitious roots.


47

A) Type of Roots, Rhizomes etc: either fresh or dry; whole or sliced; peeled or unpeeled.

B) Shape of the drug: straight, branching, tortuous, cylindrical, conical etc.

C) Surface characteristics: presence of scale leaves, cracks, scars, lenticels etc.

10.1.5 Fruits

The description of fruits shall include the class (Simple, Dry, Indehiscent fruits; Simple, Dry,

Dehiscent fruits; Schizocarpic or splitting fruits; Succulent fruits); shape and dimension; adhesion

(superior or inferior); Dehiscence (Dehiscent or indehiscent). Number of seeds and description about

organoleptic characteristics.

10.1.6 Seeds

Seeds may be produced from orthotropous, campylotropous or anatropous ovules. Care must be taken

to distinguish seeds from fruits or parts of fruits containing a single seed.

Size, Shape and Colour

Funicle

Hilum and Micropyle: Size and positions

Nature of Seed Coats

Presence or absence of perisperm and endosperm.

10.2 MICROSCOPIC EVALUATION

It involves gross microscopic examination of the drug and can be used to identify the organized/

unorganized drugs by their known histological characters. Before examination through microscope,

the material must be prepared by powdering, cutting the sections of the drug or preparing a macerate.

Crude drug can also be identified microscopically by cutting the thin TS (transverse section) / LS

(Longitudinal section). Staining reactions done on very fine sections with various staining reagents

for studies of various cellular tissues and their arrangements such as trichomes, stomata, starch

granules and calcium oxalate crystals etc. Quantitative aspects of microscopy also include study of

stomatal index, palisade ratio, vein-islet number, size of starch grains and length of fibers etc.

Examples of some of the staining reagents are:

Ferric chloride for tannin

Phloroglucinol and hydrochloric acid for lignified tissues

http://www.stafaband.info/download/mp3/lagu__ve_main_tenu_kina_chauni_hann_gal_teri/

http://www.stafaband.info/download/mp3/lagu__ve_main_tenu_kina_chauni_hann_gal_teri/


48

Ruthenium red for gums and mucilage containing cells

Chlor-zinc iodide reagent for cellulosic tissues

Acetic acid and hydrochloric acid for calcium carbonate crystals

Potassium hydroxide for hydroxyl anthraquinone.

Equipments and reagents required:

A microscope equipped with lenses with a magnification of 4x, 10x and 40x. and100

x.

A set of polarizing filters.

A stage micrometer and ocular micrometer

A heating device for preparation of sample slide

Slides and cover slip/ watch glasses.

A set of dissecting instruments.

Common Stains and Reagents for Microscopic evaluation

For the purpose of identification and characterization of materials expected to be included in the

prescribed standards, the following stains and reagents are recommended for use wherever relevant, in

addition to those mentioned in the monograph.

Acetic Acid: Dilute 6 ml of glacial acetic acid with 100 ml of distilled water; used for identification

of cystoliths, which dissolve with effervescence.

Aniline Chloride Solution: Dissolve 2 g in a mixture of 65 ml of 30 per cent ethyl alcohol and 15 ml

distilled water and add 2 ml of conc. hydrochloric acid. Lignified tissues are stained bright yellow.

Bismarck Brown: Dissolve 1 g in 100 ml of 95 per cent of ethyl alcohol; used as a general stain for

macerated material (With Schultze’s).

Chlorinated Soda Solution (Bleaching Solution): Dissolve 75 g of sodium carbonate in 125 ml of

distilled water; triturate 50 g of chlorinated lime (bleaching powder) in a mortar with 75 ml of

distilled water, adding it little by little. Mix the two liquids and shake occasionally for three or four

hours. Filter and store, protected from light. Used for lightening highly coloured material, by warming

in it and washing the tissues thoroughly.

Breamer’s Reagent: Dissolve 1 g of sodium tungstate and 2 g of sodium acetate in sufficient

quantity of water to make 10 ml. Yellowish to brown precipitates; indicate the presence of tannins.

Canada Balsam (as a Mountant): Heat Canada balsam on a water bath until volatile matter is

removed and the residue sets to a hard mass on cooling. Dissolve residue in xylene to from a thin

syrupy liquid. Used for making permanent mounts of reference slides of selected debris.


49

Choral Hydrate Solution: Dissolve 50 g of chloral hydrate in 20 ml of distilled water. A valuable

clarifying agent for rendering tissues transparent and clear, by freeing them from most of the ergastic

substances, but leaving calcium oxalate crystals unaffected.

Chloral Iodine: Saturate chloral hydrate solution with iodine, leaving a few crystals undissolved;

useful for detecting minute grains of starch otherwise undetectable.

Chlorziniciodine (Iodinated Zinc Chloride Solution): Dissolve 20 g of zinc chloride and 6.5 g of

potassium iodide in 10 ml of distilled water. Add 0.5 g of iodine and shake for about fifteen minutes

before filtering. Dilute, if needed, prior to use. Renders cellulosic walls bluish violet and lignified

walls yellowish brown to brown.

Chromic Acid Solution: 10 g of potassium chromate dissolved in 90 ml of dilute sulphuric acid: A

macerating agent similar to Schultze’s.

Corallin Soda: Dissolve 5 g of corallin in 100 ml of 90 per cent ethyl alcohol. Dissolve 25 g of

sodium carbonate in 100 ml distilled water, keep the solutions separate and mix when required, by 1

ml of the corallin solution to 20 ml of the aqueous sodium carbonate solution. Prepare fresh each

time, as the mixture will not keep for long. Used for staining sieve plates and callus bright pink and

imparts a reddish tinge to starch grains and lignidied tissues.

Ammonical Solution of Copper Oxide (Cuoxam): Triturate 0.5 g of copper carbonate in a mortar

with 10 ml of distilled water and gradually add 10 mlof strong solution of ammonia (ap.gr. 0.880)

with continued stirring; used for dissolving cellulosic materials.

Eosin: 1 per cent solution in 90 per cent ethyl alcohol; Stains cellulose and aleurone grains red.

Ferric Chloride Solution: A 5 per cent solution of ferric chloride in distilled water. Tannins

containing tissues coloured bluish or greenish black.

Glycerin: Pure or diluted as required with one or two volumes of distilled water. Used as a general

mountant.

Haematoxylin, Delafield’s: Prepare a saturated solution of ammonia alum. To 100 ml of this add a

solution of one g of Haematoxylin in 6 ml of ethyl alcohol (97 per cent). Leave the mixed solution

exposed to air and light in an unstopped bottle for three or four days. Filter and add to the filtrate 20

ml of glycerine and 25 ml of methyl alcohol. Allow the solution to stand exposed to light, till it

acquires a dark colour (about two months). Refilter and store as a stock solution. Dilute it 3 or 4 times

volumes with distilled water. Stains cellulosic fibers blue; used only on water washed material.


50

Iodine Water: Mix one volume of decinormal iodine with 4 volumes of distilled water. Stains starch

blue, and reveals crystalloids and globoids when present in aleurone grains.

Iodine in Potassium Iodide Solution: Dissolve 1 g of potassium iodide in 200 ml of distilled water,

add 2 g of iodine to the solution and dissolve it; stains lignified walls yellow and cellulosic walls blue.

Lactophenol (Amman’s Fluid): Phenol 20 g, lactic acid 20 g, glycerine 40 g, dissolved in distilled

water 20 ml; reveals starch grains in polarized light with a well marked cross at hilum, and also

minute crystals of calcium oxalate as brightly polarizing points of light.

Methylene Blue: A solution of 0.1 g of methylene blue in 25 ml of ethyl alcohol (95 per cent). A

general stain for nucleus and bacteria.

Millon’s Reagent: Dissolve one volume of mercury in 9 volumes of fuming nitric acid (Sp. gr. 1.52),

keeping the mixture well cooled during reaction. Add equal volume distilled water when cool. Stains

proteins red.

Naphthol Solution: Dissolve 10 gm of naphthol in 100 ml of ethyl alcohol; a specific stain for

detection of inulin; cells containing inulin turn deep reddish violet.

Phloroglucinol: 1 gm of phloroglucinol dissolved in 100 ml of 90 per cent ethyl alcohol; mount

debris in a few drop, allow to react for a minute, draw off excess of reagent with a filter paper strip

and add a drop of conc. hydrochloric acid to the slide; lignified tissues acquire a deep purplish red

colour; very effective on water washed material but not in chloral hydrate washed debris, for which

alcoholic solution of safranin is more effective (See safranin).

Picric Acid Solution (Trinitrophenol Solution): A saturated aqueous solution made by dissolving 1

g of picric acid in 95 ml of distilled water; stains animal and insect tissue, a light to deep yellow; in a

solution with ethy alcohol, aleurone grains and fungal hyphae are stained yellow.

Potash, Caustic: A 5 per cent aqueous solution; used to separate tenacious tissues of epidermis and

also laticiferous elements and vittae, both of which are stained brown.

Ruthenium Red: Dissolve 0.008 g of ruthenium red in 10 ml of a 10 per cent solution of lead acetate;

(to be freshly prepared) used for identification of most kinds of mucilage containing tissues, which

turn pink. A 0.0008 g ruthenium red dissolved in 10 ml of distilled water and used immediately stains

cuticular tissues in debris to a light pink.

Safranin: A one per cent solution in 50 per cent ethyl alcohol; used to stain lignified cell walls deep

red, even after clearing with choral hydrate.


51

Schultze’s Maceration Fluid: Add isolated debris to 50 per cent conc. nitric acid in a test tube and

warm over water bath: add a few crystals of potassium chlorate while warming, till tissues soften;

cool, wash with water thoroughly and tease out for mounting hard tissues; isolated cell structures are

clearly revealed, but the structures are not useful for measurement of dimensions.

Sehweitzer’s Reagent: Same as Ammoniacal Copper Oxide Solution (Cuoxam).

Sudan Red III: Dissolve 0.01 g of sudan red III in 5 ml of ethyl alcohol (90 per cent) and 5 ml of

pure glycerine; suberised walls of cork cells, and fatty material in cells are stained bright red.

Sulphovanadic Acid (Mandelin’s Reagent): Triturate one g of ammonium vandate with 100 ml

conc. Sulphuric acid. Allow the deposit to subside and use the clear liquid. This is to be prepared

fresh; useful for identification of alkaloids, particularly strychnine which turns violet in the cells

containing it.


52

11. DETERMINATION OF ASH

The total ash method is designed to measure the total amount of material remaining after ignition.

This includes both “physiological ash”, which is derived from the plant tissue itself, and “non-

physiological” ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the

plant surface.

Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and

igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand

and siliceous earth.

Water-soluble ash is the difference in weight between the total ash and the residue after treatment of

the total ash with water.

11.1 Total Ash (IP, Appendix 2.3.19)

Method A. For crude vegetable drugs

Unless otherwise stated in the individual monograph, weigh accurately 2 to 3 g of the air-dried drug in

a tared platinum or silica dish and incinerate at a temperature not exceeding 450° until free from

carbon, cool and weigh. If a carbon-free ash is not obtained, wash the charred mass with hot water,

collect the residue on as ashless filter paper, incinerate the residue and filter paper until the ash is

white or nearly white, add the filtrate to the dish, evaporate to dryness and ignite at a temperature not

exceeding 450°. Calculate the percentage of ash on the dried drug basis.

Percentage of ash with reference to air dried drug =

11.2 Acid-insoluble ash

Use Method C unless otherwise directed.

Method C. Boil the ash (Method A) with 25 ml of 2 M hydrochloric acid for 5 minutes, collect the

insoluble matter in a Gooch crucible or on an ashless filter paper, wash with hot water, ignite, cool in

a desiccator and weigh. Calculate the percentage of acid-insoluble ash on the dried drug basis.

Method D. Place the ash (Method A), or the sulphated ash (2.3.18), as directed in the individual

monograph, in a crucible, add 15 ml of water and 10 ml of hydrochloric acid, cover with a watch

glass, boil for 10 minutes, and allow to cool. Collect the insoluble matter on an ashless filter paper,

Weight of ash (g)

Weight of the drug (g)

X 100


53

wash with hot water until the filtrate is neutral, ignite to dull redness, cool in a desiccator and weigh.

Calculate the percentage of acid-insoluble ash on the dried basis.

11.3 WATER-SOLUBLE ASH

Boil the ash (Method A) for 5 minutes, with 25ml of water. Collect the insoluble matter in a gooch

crucible or on an ashless filter paper, wash with hot water and ignite for 15 minutes at a temperature

not exceeding 450°C. Subtract the weight of the insoluble matter from the weight of the ash; the

difference in weight represents the water soluble ash. Calculate the percentage of water soluble ash on

the dried basis.

Percentage of acid insoluble ash

with reference to air dried drug =

Weight of acid insoluble ash

Weight of the drug X 100

Weight of acid soluble ash

Weight of the drug X 100

Percentage of acid insoluble ash

with reference to air dried drug =


54

12. DETERMINATION OF EXTRACTIVE VALUES

This method determines the amount of active constituents extracted with solvents from a given

amount of herbal material. The extractive values by different solvents are used to assess quality,

purity and to detect adulteration due to exhausted and incorrectly processed drugs.

12.1 ETHANOL – SOLUBLE EXTRACTIVE (IP Appendix 2.6.2)

Macerate 5g of the air-dried drug, coarsely powdered into a 250ml conical flask with

stopper.

Add 100 ml of ethanol of the specified strength.

Shake the flask frequently during the first 6 hours.

Keep it aside without disturbing for 18 hours and then filter rapidly taking

precautions against loss of ethanol.

Pipette out 25ml of the filtrate and evaporate to dryness in a tared flat-bottomed

shallow dish.

Then dry at 105° and weigh.

Calculate the percentage of ethanol soluble extractive with reference to air-dried

material by the given formula:

Weight of residue

Weight of the drug

It is expressed as per cent w/w of the ‘air-dried drug’.

12.2 WATER SOLUBLE EXTRACTIVE (IP Appendix 2.6.3)

Method I-

Proceed as directed for determination of Ethanol–Soluble Extractive, using chloroform water (2.5 ml

chloroform in purified water to produce 1000 ml) instead of ethanol.

Method II-

Add 5g to 50ml of water at 80° in a stoppered flask.

Shake well and allow standing for 10 minutes.

Cool and add 2g of kieselguhr and filter.

Transfer 5ml of the filtrate to a tared evaporating dish, 7.5cm in diameter.

X 100 Percentage of ethanol soluble extractive =


55

Evaporate the solvent on a water-bath, continue drying for 30min

Finally dry in a steam oven for 2 hours and weigh the residue.

Calculate the percentage of water-soluble extractive with reference to air-dried

material by the given formula:

Weight of residue

Weight of the drug

X 100 Percentage of water soluble extractive =


56

13. LOSS ON DRYING

Loss on drying is the loss of weight expressed as percentage w/w resulting from water and volatile

matter of any kind that can be driven off under specified conditions. The test is carried out on a well-

mixed sample of the substance. If the substance is in the form of large crystals, reduce the size by

rapid crushing to a powder form.

LOD is determined by drying the sample for specified period usually 3 hours at a specified

temperature (105oC). The limit is specified on the basis of the results obtained on a reasonable

number of samples of acceptable quality. When the drying temperature is indicated by a single value

other than a range, drying is carried out at the prescribed temperature ±2°.

Method (IP Appendix 2.4.19)

Unless otherwise specified in the individual monograph, use this method.

Weigh a glass-stoppered, shallow weighing bottle that has been dried under the same

conditions to be employed in the determination.

Transfer to the bottle the quantity of sample specified in the individual monograph.

Cover it and accurately weigh the bottle and the contents.

Distribute the sample as evenly as practicable by gently sidewise shaking to a depth

not exceeding 10mm.

Dry the substance by placing the loaded bottle in the drying chamber as directed in

the monograph, remove the stopper and leave it in the chamber.

Dry the sample to constant weight or for the specified time and at a temperature

indicated in the monograph.

After drying is completed, open the drying chamber, close the bottle promptly and

allow it to cool to room temperature (where applicable) in a desiccators before

weighing.


57

14. CONTAMINANTS

Determination of foreign organic matter

Herbal materials should be free, as much as possible, of mould, insects and other animal contaminants

(animal excreta/other noxious foreign matter). Abnormal odour, discoloration, slimeness or signs of

deterioration should be absent. Amount of foreign organic matter should not be higher than the

pharmacopoeial limit.

Macroscopic examination can conveniently be employed for determining the presence of foreign

matter in whole or cut plant materials. However, microscopy is indispensable for powdered materials.

Any soil, stones, sand, dust and other foreign inorganic matter must be removed before herbal

materials are cut or ground for testing.

14.1 Definition

Foreign organic matter according to IP is material consisting of any or all of the following:

Parts of the organs or organs from which the drug is derived other than the parts named in the

definition and description or for which the limit is prescribed in the individual monograph.

Any organs other than those named in the definition and description.

Matter not coming from the source plant and

Moulds, insects or other animal contamination.

14.2 Method (IP, Appendix 2.6.1)

Weigh 100 to 500g, or quantity specified in the individual monograph, of the sample.

Spread it on a thin layer.

Inspect the sample with the unaided eye or with the use of a 6x lens.

Separate the foreign organic matter manually as completely as possible.

Weigh and determine the percentage of foreign organic matter from the weight of drug taken.

14.3 Determination of Heavy metals (IP Appendix 2.3.13). Heavy metals may be present in herbs

as a consequence of natural occurrence or from human activities such as industrial waste in the

soil; irrigation with contaminated water or airborne pollution. A general chapter on determination

of Heavy metals in herbal drugs ( Appendix 2.3.13) is included in the Indian Pharmacopoeia.


58

Example: Ashwagandha Dry Extract Monograph, IP

Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B

(20 ppm).

Methods of heavy metal determination is given below:

Method A

Standard solution. Into a 50-ml Nessler cylinder pipette 1.0 ml of lead standard solution (20 ppm

Pb) and dilute with water to 25 ml. Adjust with dilute acetic acid or dilute ammonia solution to a pH

between 3.0 and 4.0, dilute with water to about 35 ml and mix.

Test solution. Into a 50-ml Nessler cylinder place 25 ml of the solution prepared for the test as

directed in the individual monograph or dissolve the specified quantity of the substance under

examination in sufficient water to produce 25 ml. Adjust with dilute acetic acid or dilute ammonia

solution to a pH between 3.0 and 4.0, dilute with water to about 35 ml and mix.

Method B

Standard solution. Proceed as directed under Method A.

Test solution. Weigh in a suitable crucible the quantity of the substance specified in the individual

monograph, add sufficient sulphuric acid to wet the sample, ignite carefully at a low temperature until

thoroughly charred. Add to the charred mass 2 ml of nitric acid and 5 drops of sulphuric acid and heat

cautiously until white fumes are no longer evolved. Ignite, preferably in a muffle furnace, at 500° to

600°, until the carbon is completely burnt off. Cool, add 4 ml of hydrochloric acid, cover, digest on a

water-bath for 15 minutes, uncover and slowly evaporate to dryness on a water-bath. Moisten the

residue with 1 drop of hydrochloric acid, add 10 ml of hot water and digest for 2 minutes. Add

ammonia solution drop wise until the solution is just alkaline to litmus paper, dilute to 25 ml with

water and adjust with dilute acetic acid to a pH between 3.0 and 4.0. Filter, if necessary, rinse the

crucible and filter with 10 ml of water, combine the filtrate and washings in a 50-ml Nessler cylinder,

dilute with water to about 35 ml and mix.

14.4 Microbial Contamination (IP, Appendix 2.2.9)

Herbal plants and drugs are generally contaminated with a great number of bacteria and molds arising

from the soil and surrounding environment. In addition, further contamination results from harvesting

practices, handling, and processing.

A general chapter on Microbial Contamination on Non-sterile products designed for the estimation of

number of viable microorganisms present, for detecting the presence of designated microbial species


59

and acceptance criteria for herbs, processed herbs and herbal products is included in the Indian

Pharmacopoeia.

Acceptance criteria for microbiological quality are based on the Total Aerobic Viable Counts

(TAC), Total Fungal Counts (TFC) and specified pathogens.


60

15. THIN LAYER CHROMATOGRAPHY

15.1 INTRODUCTION

Thin layer chromatography (TLC) is a chromatographic technique applicable for quick screening of

herbs and herbal products by means of fingerprints. This technique is used as

1. The identity, purity, adulterations or substitutions of herbal drugs can be monitored by

comparison with the chromatogram of the standard drugs.

2. It enables entire chromatographic separation at a glance, along with semi-quantitative

information of active chemical constituents present, thus enabling assessment of quality of the

herb.

3. Optimization of TLC is fast and at a low cost by changing mobile and stationary phase.

4. TLC can be used to analyze combination of drugs with aid of appropriate separation

procedures.

15.2 BASIC PRINCIPLES OF TLC

TLC is based on the principle of adsorption, depending on the particular type of support, its

preparation and its use with different solvent. The components of the sample having more affinity

towards stationary phase migrate slowly as compared to the components having affinity towards

mobile phase. Separation in TLC is achieved by application of a solution of the sample containing a

mixture of compounds to the layer of sorbent (stationary phase), near one edge, as a spot/band on a

TLC plates of thickness 0.1-0.2 mm for analytical purposes and around 0.5-2mm for preparative TLC

are commercially available. The plate is then placed into a closed developing chamber. The solvent

front then migrates up the plate through the absorbent by capillary action.

The Rƒ is calculated by dividing the distance the compound travelled from the original position by the

distance the solvent travelled from the original position (the solvent front)

Distance travelled by sample from starting point

Distance travelled by the solvent front from starting point

15.3 Application of samples

Samples can be applied as spots or bands. Small amount of sample should be used and the sample and

reference substances should be dissolved in same solvent or solvent mixture. If the sample solution is

very dilute, several small applications in the same place should be done to allow the solvent to

evaporate between additions. Sample volume of 5-10µl should be applied on the plate, about one

Rƒ =


61

centimeter above the base level. The starting position can be indicated by making a small mark near

the edge of the plate. The plate is then dipped in to a saturated TLC chamber containing suitable

solvent. The solvent front is allowed to rise up to 80% of the plate. Plate is then dried on a suitable

heating device and then visualized and inference is drawn.

15.4 Choice of Solvent system

The solvent system should be chosen to match the nature of analytes and sorbent being used. Because

the mobile phase competes with the sample for sorbent sites, polar substances require a polar solvent

to migrate on a silica gel or alumina adsorbent layer. In reverse-phase TLC, non-polar substances are

strongly attracted to the layer, and non-polar mobile phases are required to effect migration.

Some of solvent systems used for separation of herbs and herbal drugs have been shown in Table 1.

Table 1. Solvent systems used for development of TLC chromatogram of phytoconstituents


62

SOLVENT SYSTEMS SOLVENT RATIO SORBENTS CHEMICAL

CONSTITUENT

Essential Oils

Toluene: Ethyl acetate 93:7 Silica gel 60F254 Anethole/ methylchavicol

or safrole, eugenol

Toluene - Silica gel 60F254 Eugenol

Toluene:Ethyl acetate 93:7 Silica gel 60F254

Carvone, Linalool,

Cineole, α – terpineol,

thymol, carvacrol

Toluene: Ethyl acetate 93:7 Silica gel 60F254

Menthol, menthone,

methyl acetate,

menthofuran

Alkaloidal Drugs

Toluene: Ethyl acetate :

Diethylamine 70:20:10 Silica gel 60F254

Suitable for major

alkaloids of most drugs

Ethyl acetate: Methanol:Water 100:13.5:10 Silica gel 60F254

Xanthine derivatives,

colchicum and Rauwolfia

alkaloids

Acetone: Water: conc.

Ammonia 90:7:3 Silica gel 60F254

Solanaceous, Atropine and

Hyoscyamine alkaloids

Chloroform: Diethylamine 90:10 Silica gel 60F254 Cinchona alkaloids

Toluene: Acetone: Ethanol :

conc. Ammonia 40:40:6:2 Silica gel 60F254

Opium alkaloids

n-Heptane: Ethylmethyl

ketone: Methanol 58:34:8 Silica gel 60F254

Rauwolfia alkaloids

Cyclohexane: Chloroform :

Glacial acetic acid 45:45:10 Silica gel 60F254

Berberine and

protoberberine type

alkaloids

Bitter Drugs

Ethyl acetate: Methanol:

Water 77:13.5:8 Silica gel 60F254

General system for

screening

Acetone: Choloroform: Water 70:30:2 Silica gel 60F254 Amarogentin

Chloroform: Methanol 95:5 Silica gel 60F254 Quassin

Iso-octane: Isopropanol:

Formic acid 83.5:16.5:0.5 Silica gel 60F254

Humulone and lupulone

n- heptanes: Isopropanol:

Formic acid 90:15:0.5 Silica gel 60F254

Bitter acids

Chloroform: Ethanol 95:5 Silica gel 60F254 Cucurbitacins


63

Anthracene derivatives


Water 100:13.5:10 Silica gel 60F254

All anthracene drug

extracts

n-propanol: Ethyl acetate:

Water: Glacial acetic acid 40:40:29:1 Silica gel 60F254

Senna

Light petroleum: Ethyl acetate

:Formic acid 75:25:1 Silica gel 60F254

Anthraquinone aglycones

Toluene: Ethylformate:

Formic acid 50:40:10 Silica gel 60F254

Hypericin

Cardiac Glycosides


Water

100:13.5:10

Or

81:11:8

Silica gel 60F254 Applicable system for

cardiac glycosides


Ethanol: Water 81:11:4:8 Silica gel 60F254

Strophanthoside. Addition

of ethanol increases Rf

value of strongly polar

compound

Chloroform: Methanol: Water 64:50:10 Silica gel 60F254 Separation of all saponin

mixtures from plants.

Flavonoids

Ethyl acetate: Formic acid:

Glacial acetic acid: Water 100:11:11:26 Silica gel 60F254

Screening system for

flavonoid glycosides


Glacial acetic acid:

Ethylmethyl ketone: Water

50:7:3:30:10 Silica gel 60F254 Screening system for

flavonoid glycosides

Benzene: Pyridine: Formic

acid 72:18:10 Silica gel 60F254 Flavonoid aglycones

Chloroform: Acetone: Formic

acid 75:16.5:8.5 Silica gel 60F254 Flavonoids

Toluene: Ethylformate:

Formic acid 50:40:10 Silica gel 60F254 Flavonoid aglycones

Coumarins

Toluene: Ether (1:1) saturated

With 10% acetic

Acid

Silica gel 60F254 Universally accepted for

coumarin aglycones

Ethyl acetate - Silica gel 60F254 Used for higher Rf


64

Source: In Mukherjee PK (2002). Thin Layer Chromatography. Quality Control of Herbal Drugs. Business Horizons publisher. 1st edition.

15.5 Derivatization and Detection of TLC plate

Zone detection in TLC is based on natural colour, fluorescence, or UV absorption or on the use of

various universal or selective chemical detection reagents applied by spraying or dipping. Detection

with UV radiation at wavelengths 254 nm and/or 366 nm is fast and straight forward detection for

visualization of separated substances. After the plate is developed, it is sprayed with various reagents

for the development of colour. Often the colour reaction is not confined to a single compound but is

produced by several compounds belonging to a particular group. Some of the colouring reagents

commonly used for analysis of herbal drugs is listed in Table 2 wherein the correlations of colour

response with particular aspect of drug structure are reviewed.

Table 2: Most common derivatization reagents

Vanillin/Sulfuric acid A universal spray, many terpenoids, sterols and

saponins give red and blue colours.

Ammonia vapour Alkaloids, Flavonoids, mycotoxins, anthracene

Aniline-diphenylamine- phosphoric acid

Phenols, Flavonoids, tannins, plant acids, ergot

alkaloids, hypericin.

Anisaldehyde/ vanillin with sulphuric or

phosphoric acid

Steroids, higher alcohols, phenols, and

essential oils.

Bromocresol Green Solution Detection of flavonoids.

Dragendorff’s reagent Alkaloids, heterocyclic nitrogen compounds

and lipids. Alkaloids give dark orange or red


Glacial acetic acid: Water 100:11:11:27 Silica gel 60F254 For polar coumarins

Lignans

Chloroform: Methanol (6cm)

followed by toluene-acetone

90:10 followed by

65:35 Silica gel 60F254 Podophyllins

Pungent principles from plants

Toluene: Ethylacetate 70:30 Silica gel 60F254 Piper

Toluene: Ethyl ether: Dioxan 62.5:21.5:16 Silica gel 60F254

General for pungent

principles

Diethyl ether (saturated

chamber) - Silica gel 60F254

Capsaicin and related

compounds

Triterpenes

Ethyl acetate: Glacial acetic

acid: Formic acid: Water 100:11:11:26 Silica gel 60F254

Polar compounds

(isoflavone)


65

coloration.

Fast blue salt Tannins, coumarins, cannabinoids, amines.

Ferric (III) chloride Phenolic compounds for example, coumarins,

flavonoids, tannins. Blue or red coloration will

be observed.

Iodine vapour For many organic compounds

Ninhydrin Amino-acids, Primary amines, secondary

amines and peptides.

Phosphomolybdic acid solution For detection of alkaloids

15.6 Troubleshooting in TLC analysis

When compound runs as a streak rather than a spot, due to overloading. Run the TLC again

after diluting your sample. Or, sample might contain many components, creating many spots

which run together and appear as a streak. Perhaps, the experiment did not go as well as

expected.

When the sample runs as a smear or an upward crescent. Compounds which possess strongly

acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with

this behaviour. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic

acids) to the eluting solvent to obtain clearer plates.

If the sample runs as a downward crescent, the adsorbent may be disturbed during the

spotting, causing the crescent shape.

When the plate solvent front runs crookedly. Either because of the adsorbent has flaked off

the sides of the plate or the sides of the plate are touching the sides of the container (or the

paper used to saturate the container) as the plate develops. Crookedly run plates make it

harder to measure Rf values accurately.

Many random spots are seen on the plate, indicating analyst has accidentally dropped any

organic compound on the plate.

No spots are seen indicating solvent level is above baseline.


66

16. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

16.1 INTRODUCTION

High Performance Thin Layer Chromatography (HPTLC) is also known as planar chromatography.

This analytical technique is known for its high separation capability, performance and reproducibility

considered superior to classical TLC methods. HPTLC have been widely used for analysis of Herbs

and Herbal Products. Although, TLC is a simple, rapid method but it has many pitfalls. HPTLC came

into existence in 1975 with introduction of high efficiency, commercially precoated plates, that are

smaller (10 x 10 or 10 x 20 cm), have thinner (0.1- 0.2 mm) layer composed of sorbent with a finer

mean particle size (5-6µm) and a narrower particle size distribution or classification (4-8 µm) and are

developed over shorter distances (about 3-7 cm) compared to classical TLC plates, which are 20 x 20

cm with a 0.25 mm layer containing particles with an average size of 10-12 µm.

HPTLC is a flexible, versatile, economical process in which all the processes are independent. Since

herbs are complex mixtures and herbal extracts contain numerous compounds in different

concentrations, the analysis of herbs and herbal preparations is challenging. The advantages of

HPTLC include the following:

Non tedious sample preparation technique and multiple samples can be analyzed on one plate.

Great range of stationary phases available with unique selectivity for mixture components.

Ability to choose solvents for the mobile phase is not restricted by low UV transparency or the

need for ultra-high purity. Corrosive and UV-absorbing mobile phases can be employed.

No prior treatment for solvents like filtration and degassing.

Chromatographic conditions can be changed within a few minutes, and chromatographic chamber

requires little time for equilibration.

Multiple detection of the sample chromatogram is possible in order to improve selectivity and

confirm zone identity.

Automated sample application takes 0.5-2 min per sample depending on the size, application mode

and number of replicates.

Densitometry evaluation of plates can be accomplished within 10 minutes.

The results can be easily monitored, documented and the chromatograms of various herbs and

herbal preparations can be compared.

Amount of mobile phase required is small, minimizing the cost.

Accuracy and precision of quantification is high because samples and standards are

chromatographed and analyzed under same conditions on a single plate.


67

HPTLC is a modern adaptation of TLC with better and advanced separation efficiency and detection

limits. The table below compares HPTLC and TLC (Table 1).

Table.1: Comparison of TLC and HPTLC

SNO. FEATURES TLC HPTLC

1 Technique Manual Semiautomatic

2 Layer Lab made/ Precoated Precoated

3 Sample

application Circular Rectangular or Circular

4 Solid support Silica gel , Alumina &

Kieselguhr etc..

Wide choice of stationary phases

like silica gel for normal phase and

C8 , C18 for reversed phase modes

etc

5 PC

connectivity No Yes

6 Method

storage No Yes

7 Validation No Yes

8 Quantitative

analysis Difficult Relatively easier

9 Sample holder Capillary/ Pipette Syringe

10 Wavelength

range 254 or 366 nm, visible 190 to 800nm, monochromatic

11 Analysis Time Slower Shorter migration distance and the

analysis time is greatly reduced

12 Scanning Not possible

Use of UV/ Visible/ Fluorescence

scanner scans the entire

chromatogram qualitatively and

quantitatively and the scanner is an

advanced type of densitometer

13 Accuracy Low High

16.2 HPTLC METHOD DEVELOPMENT

The method for analyzing herbals by HPTLC demands primary knowledge about the nature of

chemical constituents present in herbs. In combination with microscopic investigations, they provide a

means for identity check. Any adulterations in raw material can also be identified. Method

development and standardization involves trail and errors. The first step is the selection of appropriate

developing solvent. This can be done by, identifying the solvents that can have average separation

power for the desired constituent(s), employing a gradient system and use of modifiers like acids or

bases. Selection of a marker compound(s) for a particular herb or herbal preparation(s) for

quantification is also of critical importance. It is often impossible to separate all the components of the

plant/extract. Therefore, the method shall be designed such that the marker compound doesn’t co-


68

elute with other substances. Procedure for HPTLC method development is outlined as follows

(Figure1) :

FIGURE 1 Schematic procedure for HPTLC method development

16.2.1 Stationary phase

HPTLC plates comprises of small particles with a narrow size distribution resulting in homogenous

layers with a smooth surface. HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly

decreased development distance (3-7cm) and analysis time (7–20 min). Commercial precoated plates

with glass or aluminium foil backing have been extensively used. Normal phase adsorption TLC on

silica gel, usually silica gel 60 (60 Å pore size), with a less polar mobile phase, such as chloroform–

methanol, has been used for more than 90% of reported analysis. Reversed-phase lipophilic C-18

chemically-modified silica gel phase; and hydrocarbon- impregnated silica gel plates developed with

a more polar aqueous mobile phase, such as methanol–water or dioxane-water, are used for reversed-

phase LC.

Other precoated layers that are used include aluminum oxide, magnesium silicate, magnesium oxide,

polyamide, cellulose, kieselguhr, ion exchangers, and polar modified silica gel layers that contain

bonded amino, cyano, diol, and thiol groups. HPTLC plates need to be stored under appropriate

conditions. Before use, plates should be inspected under white and UV light to detect damage and

impurities in the adsorbent. It is advisable to prewash the plates to improve the reproducibility and

robustness of the results.

Test solution and Reference solution Selection of chromatographic plate and mobile phase

Plater pre-washing if required

Saturation of chromatographic chamber with mobile

phase

Application of test and reference solution followed by drying of spotted plate

Chromatographic development followed by drying of spotted plate

Detection of spots

Scanning & Documentation of developed plate

Archiving


69

16.2.2 Mobile Phase

The selection of mobile phase is based on adsorbent material used as stationary phase and physical

and chemical properties of analyte. Generally diethyl ether, methylene chloride, and chloroform

combined individually or together with hexane as the strength-adjusting solvent for normal-phase

TLC and methanol, acetonitrile, and tetrahydrofuran mixed with water for strength adjustments in

reverse-phase TLC are being used. Accurate volumetric measurements of the components of the

mobile phase must be performed separately and precisely in adequate volumetric glassware and

shaken to ensure proper mixing of the components.

16.2.3 Sample preparation and application of Spot

After preparation of mobile phase, the test solution is prepared by Infusion, Decoction, Maceration,

Soxhletion and Percolation technique. The amount of Test sample and Reference sample to be taken

for testing should be as specified in the monographs concerned..

A good solvent system is one that moves all the components of the mixture off the baseline, but does

not move them near to the solvent front. The spots /peaks of interest should be resolved between Rf

0.15 and 0.75. The more non-polar the compound, the faster it will elute (or the less time it will

remain on the stationary phase) and the more polar the compound the slower it will elute (or more

time on the stationary phase). The volume of reference solutions and test material may be applied to

the plate in the range of 5µl- 20µl so that when the same is applied as a band of width of

6mm-8mm, it provides reasonable amounts of the phytochemicals being aimed for separation-neither

too low nor too high proportions. . The narrow, homogenous sample bands lengths are usually applied

by use of an automated applicator. These ensure high-resolution separations, accurate and precise

quantitative results by scanning densitometry.

16.2.4 Chromatogram Development

A classical method of linear, ascending development in a mobile phase vapour-saturated, covered

glass chamber or tank is widely used for herbal analysis. The twin trough chamber is also used; it is a

normal chamber with inverted V-shaped ridge on the bottom dividing the chamber into two sections.

This allows use of low volume of mobile phase and easy conditioning of the layers. Complex

separations can be performed by use of two–dimensional development, in which the sample mixture is

applied to one corner of HPTLC plate. The plate is then developed with first mobile phase, dried and

then developed with the second mobile phase in the perpendicular direction. In addition to the usual

capillary flow development of plates, forced flow overpressure layer chromatography (OPLC) in

which the mobile phase is mechanically pumped through the layer covered with a membrane that is

under pressure have been used.

16.2.5 Developing the Plate


70

After the plate is developed, it is dried in fume hood or oven to completely evaporate the mobile

phase. The separated compounds are detected by their natural colour, natural fluorescence under 366

nm UV light, quenching of fluorescence under 254 nm UV light on an “F” layer containing a

phosphor termed as UV indicator sprayed with various reagents for the development of colour often

the colour reaction is not confined to a single compound but is produce by several compounds

belonging to a particular group. Therefore along with using migration rates, various constituents of a

sample are also identified their response to chemical treatment. Universal or selective chromogenic

and fluorogenic detection reagents are applied by spraying onto the layer, dipping the layer into

reagent, or exposing the layer to reagent vapours. An important advantage of offline operation of

HPTLC is the flexibility to use multiple detection methods. For example, the layer can be viewed

under long wave, short wave UV light, followed by one or more chromogenic, fluorogenic or

biological detection methods. The most common derivatizing agents are enlisted in chapter of TLC.

16.2.6 Quantification

The quantitative evaluation is performed by measuring the predefined zones/spots obtained in the

samples and standards using a densitometer or scanner. The scanner and densitometers need to be set

to the desired UV wavelengths.

16.2.7 Documentation

Documentation to be done with the help of controlled automated system and help of software. While

doing so they need to be suitably numbered and a numbering system need to be developed and

adopted for proper storage of the documents/profiles/densitometry data and also for easy retrieval of

them.

16.3 Fingerprint of some medicinal plants by TLC/HPTLC method

SNO. HERBAL DRUGS (IP

2014)

CATEGORY MOBILE PHASE SPRAY

REAGENTS

1. Trachyspermum ammi

(Ajwain)

Antiasthmatic,

carminative,

germicidal,

antioxidant

Toluene : ethyl acetate

[93:7]

Anisaldehyde

sulphuric acid

reagent

2 Emblic myrobalan

(Amalaki)

Antiscorbutic, antacid,

carminative,

hepatoprotective


: glacial acetic acid:

formic acid

[20:45:20:5]

Anisaldehyde

sulphuric acid

reagent

3 Cassia fistula

(Amaltas)

Purgative, diuretic,

antipruritic, febrifuge


: formic acid :

methanol [3:3:0.8:0.2]

-


71

4 Mangifera indica

(Amra)

Cardiotonic,

astringent, nourishing

tonic, in haemoptysis

Ethyl acetate : formic

acid : acetic acid :

water [100:11:11:25]

Vanillin glacial

acetic acid reagent

5 Terminalia arjuna

(Arjuna)

Antihyperlipidaemic,

antihypertensive,

astringent,

cardioprotective


: acetic acid [5:5:0.5]

10% w/v sulphuric

acid in methanol

6 Atropa belladonna

(Belladona dry extract) Anticholinergic

Acetone : water :

strong ammonia

solution [90:7:3]

Modified potassium

iodobismuthate

solution

7 Asparagus racemosus

(Shatavari)

Anti-inflammatory,

immunomodulatory,

neuroprotective,

antidysentery

Chloroform : methanol

: water [13:10:2]

Vanillin sulphuric

acid reagent

8 Cassia angustifolia

(Senna)

Purgative,

anthelmintic,

febrifuge

n-propyl alcohol :

ethyl acetate : water :

glacial acetic acid

[40:40:29:1]

20% v/v of nitric

acid solution

9 Centella asiatica

(Mandukaparni)

Brain tonic, anabolic,

anxiolytic, alterative

Chloroform : glacial

acetic acid : methanol :

water [60:32:12:8]

Anisaldehyde

sulphuric acid

reagent

10 Adhatoda vasica

(Vasaka)

Expectorant,

bronchiodilator

Ethyl acetate :

methanol : strong

ammonia solution

[8:2:0.2]

Dragendorff’s

reagent

16.4 TROUBLESHOOTING IN HPTLC ANALYSIS

A poor band quality is observed due to variation in the gas flow, wrong distance between needle

tip and TLC layers or because of clogged needle. Check gas pressure and adjust to 2-5 bars,

check distance and adjust to 1mm, washing of the syringe.

Bad reproducibility due to sample overloading. Dilute the sample and inject.

Poor accuracy may be due to destroyed glass barrel of the syringe or damaged spray head due to

wrong sample dosage syringe. Replace the spray head.

The quality of bands can also be affected because of clogging in the nozzle, this requires cleaning

of the nozzle.


72

16.5 VALIDATION

The suitability of any analytical procedure for its intended use in pharmaceutical analysis must be

based on objective validation data. Guidelines on the validation of analytical procedures are available

on the website of International Conference on Harmonization (ICH) www.ich.org

TLC/HPTLC, is one of the predominant techniques for identity, quantification and standard limit

tests for impurities, especially for those that are difficult or impossible to detect with liquid

chromatography. The process of validating a procedure cannot be separated from its development as

the analyst will not know whether the procedure and its performance are acceptable until validation

has been performed. Before outlining the validation protocol and the experimental design, it is

necessary to qualify all instrumentation and equipment. All chemicals, TLC plates, and reference

standards must be defined, specified, and tested. The analytical procedure must be developed,

optimised, and documented. A validation protocol, which includes the acceptance criteria and the

specified statistical approaches, must be agreed upon and signed.

A quantitative TLC purity test requires a comprehensive validation study - including specificity,

linearity and range, precision, accuracy, detection limits, quantitation limits etc.

16.5.1 Specificity

To demonstrate specificity, the sample, standard substance(s), pure and spiked placebos are

chromatographed simultaneously. The chromatographic method selectively separates the active

constituent from the impurities and excipients.

16.5.2 Linearity and Range

Linearity is the functional relationship between the concentration of the substance and the measured

value. Ideally the calibration curve should be linear that should pass through the origin when tested by

regression analysis. Unfortunately, numerous analytical techniques indicate/demonstrate or lead to a

nonlinear relationship between the concentration of analytes in samples and the corresponding

measurement signals. In TLC/HPTLC especially in the case of evaluation performed by scanning in

the UV/VIS reflection mode, most calibration functions are nonlinear. The absorption measurement

always yields 2nd degree functions. Therefore, for routine analytical procedures, a three-point

calibration model must be used.

16.5.3 Precision

Precision is the closeness of agreement between the values obtained in an assay. It is expressed as the

coefficient of variation (% CV). CV is the standard deviation of the assay values divided by the

concentration of the analyte. Several types of precision can be measured: intra-assay precision

(repeatability) is the % CV of multiple determinations of a single sample in a single test run.

Repeatability provides information about the variation caused by sample preparation, sample


73

application, and evaluation within one analytical run and within a short period of time. The same

sample is determined on the same plate by applying at least six analytical solutions from individual

weighings. Inter-assay precision (also called intermediate precision) measures the % CV for multiple

determinations of asingle sample, controls and reagents analyzed in several assay runs in the same

laboratory. Reproducibility is the precision between different laboratories and is done on the same

sample with different apparatus.

16.5.4 Accuracy

Accuracy is the closeness of agreement between the actual value of the drug and the measured value.

Spike and recovery studies are performed to measure accuracy. The analyte is spiked with three

different concentrations of the impurities present. The recovery is calculated as a percentage value of

the added amount of impurity.

% Recovery = (Amount found/ Amount added)* 100

16.5.5 Limit of Detection/ Limit of Quantification

Limit of Detection (LOD) is the lowest amount of the analyte in a sample that can be detected but not

necessarily be quantified as an exact concentration or amount. Limit of Quantitation (LOQ) is the

lowest amount of an analyte that can be measured quantitatively in a sample with acceptable accuracy

and precision. For determination of LOD and LOQ the impurities and the analyte are run in triplicate.

A blank is also run simultaneously. Thus, LOD and LOQ are calculated based on the signal-to-noise

ratio.

Conclusion

Thus HPTLC methods are employed to analyze herbs and herbal preparations for the qualitative

(fingerprint) and quantitative determination, demonstrating their feasibility in the quality control of

phytoconstituents.


74

17. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

17.1 Introduction

HPLC is a chromatographic technique widely used for qualitative and quantitative analysis of organic

compounds present in multi-component mixtures, such as herbal plants extracts. It has a broad range

of application as it can be applied to herbal products that contain components such as alkaloids,

glycosides, flavanones, organic acids, phenols and lignans. The main HPLC conditions include the

optimal choice of column, mobile phase and detector. The HPLC conditions should be optimized, so

that the components of the herbals can be resolved as much as possible. In other words, the more

chromatographic peaks obtained, the better it is. This allows the internal characteristics of the herbal

plants to be fully manifested, providing sufficient information for the fingerprint evaluation and for its

quality assessment.

For the separation of crude extracts, either raw mixtures or samples enriched by extraction via simple

solid-phase extraction or liquid-liquid extraction are injected into HPLC after passing through 0.45µm

filter. The separations are performed mostly in reverse-phase chromatography on C18 material with

the ACN-H2O or MeOH-H2O solvent system in gradient elution mode.

17.2 Normal and Reverse Phase Chromatography

In normal phase the polarity of the stationary phase is higher than that of the mobile phase. In Reverse

phase, the polarity of stationary phase is less than that of mobile phase. For ionizable solutes, the pH

of the mobile phase is an important factor in control of retention and selectivity. Figure 1 describes

the methods for choice of HPLC methods.


75

17.3 HPLC COLUMN

MW

>20

00

M

W<2

00

0

Ionic (including

metal

chelates and

transition

metals)

Normal

phase(bonded

)

Water

Soluble

Organic

Soluble

Organic

Soluble

Chiral Sample

Hexane

Soluble

Normal

phase(adsorpt

ion)

Unmodified

silica packing

CN, NH2 bonded

phases

Non-ionic

MeOH,

MeOH/H2O

Soluble

THF

Soluble

Reverse

phase bonded

C18, C8, phenyl,

C4 , CN

bonded phases

Small

molecule GPC

Hydrophobic

small pore

packing’s

Reverse

phase(bonded)

C18, C8, phenyl,

C4 , CN

bonded phases

Reverse phase (with

ionizationcontrol)

C18, C8, phenyl, C4 , CN

bonded phases

(with pH control)

Reverse phase

(paired ion)


bonded phases

(with pH control)

Ion exchange Anion and cation

exchange columns

Water

Soluble

Chiral

chromatography

Chiral phases (eg. Pirkle

columns)

Reverse phase

(with

ionizationcontrol)


bonded phases

(with pH control)

Reverse phase

(paired ion)


bonded phases

(with pH control)

Ion exchange Anion and cation

exchange columns

Chiral

chromatography


columns)

Chiral

chromatography


columns)

Fig 1. Choice of HPLC method


76

HPLC column consists of a metal cylinder packed with tiny silica or modified silica particles to

separate compounds in a mixture. The mobile phase passes through the column using high pressure,

resulting in each component in the mixture coming out at different times, and the elution is based on

the polarity (Table 1 ).

Table1 : Properties of solvents commonly used in HPLC.

S.No. Solvent Polarity Miscible with

water

UV cut off

1 Hexane Nonpolar No 200

2 Carbon

tetrachloride

Nonpolar No 263

3 Chloroform Nonpolar No 245

4 Methylene chloride Nonpolar No 235

5 Tetrahydrofuran Nonpolar Yes 215

6 Diethyl ether Nonpolar No 215

7 Acetone Nonpolar Yes 330

8 Ethyl acetate Nonpolar Poorly 260

9 Acetonitrile Nonpolar Yes 190

10 Isopropanol Nonpolar Yes 210

11 Methanol Nonpolar Yes 205

12 Water Polar Yes -

For the herbal products that contain alkaloidal compounds, the ion exchange column may be selected.

For those that contain mainly polysaccharides compounds, a gel column may be selected. For those

that contain mainly steroidal compounds, the C18 reverse-phase column may be selected. Currently

most commonly used columns are C18 bonded-phase type of reverse-phase ODS column. The normal-

phase column, which is rarely used, is for the separation of homologous or isomeric compounds. The

ion exchange column is used for the separation of water-soluble ionic compounds. Some compounds

may require the use of amino compounds.

17.3.1 Selection of HPLC Column

The selection of HPLC columns is based on following criteria:

a) Internal Diameter

An important parameter of a HPLC column is the internal diameter (ID) as this directly influences the

Detection, sensitivity, selectivity of separation and the quantity of analyte that can be loaded onto the

column


77

Low ID columns have higher sensitivity and lower solvent consumption for analytical

purposes and require low loading of sample

Analytical scale columns (4 mm and 4.6 mm) are the most common type of columns and

often use a UV‐Vis absorbance detector.

Narrow‐bore columns with ID of ≤ 3mm are used for sensitive analytical applications and use

more advanced UV‐Vis, fluorescence or mass detectors

Large ID columns (≥ 7mm) have a high loading capacity and so are used to purify usable

amounts of sample material for preparative or semi‐preparative applications

Internal diameter of the column has vital role in resolution and is inversely proportional i.e.

lesser the internal diameter better the resolution.

b) Particle size

The size of the particles (beads) in the stationary phase of the column influences the separation of

the analyte. Smaller particles generally provide more surface area and better separations, but the

pressure required for optimum separation is greater.

Columns with 5and 3μm are commonly used. Columns with 5 μm particles provide high

resolution of peaks and are large enough to be suitable for analytical, preparative and

semi‐preparative applications and with 3μm particles are used when rapid analysis is needed and

are mostly suitable for analytical applications

c) Porosity

An ideal stationary phase will be porous enough to provide greater surface area so that the sample

material can fit into the column and be analysed. Small pores up to 120 Å provide greater surface

are a and lead to higher resolving ability and are typically used for smaller MW analytes. Larger

pores such as 300 Å are suitable for a wider range of samples especially for larger MW analytes

such as proteins and oligonucleotides.

d) Length. The length of a column determines the overall separation time and influences the

resolution of the peaks. For the same particle size:‐

3 ‐ 7.5cm length column provide faster analysis run times, however these may be relatively

costlier

10 – 25cm length columns provide better resolution and are commonly used.


78

A compromise between the speed and resolution of analysis is normally needed to find the most

efficient column i.e. the shortest column with the best resolving capability.

e) Chemical nature of compounds

If compounds have many carbons, or differ in the number of carbons, choose a stationary

phase with carbon (C18, C8, and phenyl). This works well for molecules that are soluble in

solvents such as acetonitrile, methanol, etc.

Acids and bases can be difficult to separate. The "neutral" form is usually retained more on a

reversed phase (C18) column, while the "ionic" form is not retained as much. Control of pH

is required in such cases.

A phenyl column for aromatic compounds separation and amino and -Cyano columns can be

used for separating polar organic molecules such as sugars.

17.4 MOBILE PHASE

The choice of mobile phase depends on the nature of the drug and the solvent system with the most

optimized method should be used. The best separation should satisfy the following requirements:

All the components of the test sample can be analyzed or the sample can be analyzed into as

many components (peaks) as possible.

The peaks should be adequately resolved.

The analysis time is as short as possible.

Optimization of the separation conditions mainly involves optimization of chemical factors such as

mobile phase composition, pH value of the mobile phase and ion pair reagent concentration.

The separation may be achieved either in isocratic mode or gradient mode. As the herbal drugs

contain complex components, normal isocratic elution is unable to achieve the separation of

components of different natures, giving rise to fewer peaks in the chromatogram and insufficient

information required for fingerprint evaluation. Under most circumstances, gradient elution should be

used. Under suitable gradient conditions, compounds of widely differing natures are all separated.

17.5 DETECTORS

The detectors available for detection of herbal compounds include ultraviolet-visiblet (UV-Vis)

detector - diode array detector (DAD), fluorescence detector (FLD), electrochemical detector (ECD)

and refractive Index detector (RID).

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79

17.5.1 UV DETECTOR

It is suitable for detection of compounds with ᴫ -ᴫ conjugated or ᴩ -ᴫ conjugated structure, while a

large number of compounds that are without double bonds are not detected. Three types of UV

detectors are available: fixed wavelength, multiple wavelength or photodiode array (DAD). The fixed

wavelength detector is the least expensive and has higher intrinsic sensitivity because the light is

emitted at specific wavelengths with given lamps. However, the multiple-wavelength detector is more

versatile and can compensate for its lower sensitivity when a wavelength with the higher extinction

coefficient for the solutes is chosen. UV-visible detector has its application in detection of flavonoids,

terpenes, alkaloids, coumarins and alkamides.

17.5.2 FLUORESCENCE DETECTOR (FD)

Compared with HPLC-UV, fluorescence detector (FD) affords much greater sensitivity and

selectivity. In fluorescence, the molecular absorption of a photon triggers the emission of another

photon with a longer wavelength. This difference in wavelength provides more selectivity and the

fluorescent light is measured against a very low light background, thus improving the S/N ratio. Most

application of FD is related to detection of aflatoxins in food because they contain natural

fluorescence.

17.5.3 REFRACTIVE INDEX DETECTOR (RID)

RID is a simple and least expensive detector. The detector has been useful for detecting compounds

such as carbohydrates and polymers. However, RID lacks sensitivity and is susceptible to changes in

ambient temperature, pressure, and flow rate and cannot be used for gradient elution.

17.6 SAMPLE ANALYSIS

The herbal plants are prepared using optimized extraction and separation methods, and the analysis

should be carried out under the best chromatographic conditions for separation and analysis. Rigorous

method validation should be done to confirm the reliability of the test results, comprising mainly of

precision of the instrument and the reproducibility of the test method.

17.7 SYSTEM SUITABILITY

A system suitability test is required to ensure that a given operating system may be generally

applicable. System suitability also verifies that the resolution and reproducibility of the

chromatographic system are adequate for the analysis to be done.

Replicate injections of a standard solution used in the assay or other standard solution are compared to

determine whether the requirements for precision are met. Unless otherwise specified in the individual


80

monograph, data from five replicate injections of the analyte are used to calculate the relative standard

deviation if the requirement is 2% or less; data from six replicate injections are used if the relative

standard deviation is more than 2%.

Replicate injections of the standard solution may be made before the injection of samples or may be

interspersed among sample injections. System suitability must be demonstrated throughout the run by

injection of an appropriate control preparation at appropriate intervals, including at the end of the

analysis. The control preparation can be the standard solution used in the test or a solution containing

a known amount of analyte.

17.8 TROUBLESHOOTING IN HPLC

Peak Problems

No peaks usually indicate instrumental problems. It could be due to a number of

causes including no sample injected, system not turned on properly, major leaks, a

dead detector, wrong mobile phase, or a particularly retentive or adsorptive column.

No flow usually indicates pump problems, flow system, blockages, or empty solvent

reservoirs.

Too many peaks occur from sample carryover, usually in the auto sampler or on the

column. Using a sample solvent stronger than the mobile phase may cause sample

precipitation in the column. The next blank run will show original chromatogram,

but at a lower concentration.

Too few peaks indicates poor resolution i.e. one or more peak overlap each other.

Increase the resolution or use “peak purity” software with a PDA to confirm

presence of unresolved peaks.

Fronting and tailing peaks: Fronting peaks are caused by column overload so dilute

the sample 1/10 and re-inject. Tailing peaks are caused by secondary interactions of

the analyte with the stationary phase or of excess void volume. All fittings and

tubing’s between the injector and the detector should be checked.

Broad Peaks and Split Peaks: These are indications of degraded column

performance caused by sample contamination, a partially blocked inlet frit, or

column voids caused by dissolution of silica particles usually at higher pH. Thus, the

column needs to be replaced.

Changes in Retention Time


81

Changes in flow rate and mobile phase composition are the major factor responsible for changes in

retention time. Always prepare and measure pH in the aqueous portion of the mobile phase before

adding the organic solvent. Organic solvent changes pH.

Peak area problems

Peak area changes may be due to changes in volume injected, flow rate, wavelength of detector, pH,

leaks, sample stability, integration problems and partial loss of sample due to irreversible adsorption

on a dirty frit or inactive column.

Baseline drift

Column temperature fluctuation (even small changes cause cyclic baseline rise and

fall; most often affects refractive index and conductivity detectors, or UV detectors

at high sensitivity or in direct photometric mode). The column and mobile phase

temperature should be controlled.

Non-homogeneous mobile phase (drift usually to higher absorbance, rather than

cyclic pattern from temperature fluctuation). HPLC-grade solvents should be used

and the mobile phase should be degassed before use.

Contaminant or air buildup in detector cell. Flush the cell with methanol or other

strong solvent or if necessary clean cell with 1N HNO3 (never with HCl).

Mobile-phase mixing problem or change in flow rate. Composition/flow rate should

to correct.

Mobile phase contaminated deteriorated, or prepared from low-quality materials.

Detector (UV) is not set at absorbance maximum but at slope of the curve. The

wavelength should be set at UV absorbance maximum.


82

18. ANALYSIS OF ESSENTIAL OILS

18.1 INTRODUCTION

An essential oil is primarily a substance comprising of volatile compounds which are formed through

the plant metabolism. They are isolated by physical means only (pressing and distillation) from a

whole plant or plant parts of known taxonomic origin, primarily composed of terpenes and their

oxygenated derivatives and are obtained by steam distillation or solvent extraction of different parts of

the aromatic plants including the buds, flowers, leaves, seeds, roots, stems, bark, wood and rhizomes

etc. Generally essential oils are immiscible with water, many of which are sufficiently soluble in

water to impart to its characteristics odor and taste. The two principal circumstances determine a plant

to be used as an essential oil plant:

A unique blend of volatiles like flower scents in rose. Such flowers produce and

immediately emit the volatiles by the epidermal layers of their petals.

Secretion and accumulation of volatiles in specialized anatomical structures such as

secretory idioblasts, cavities/ducts, or glandular trichomes. This leads to higher

concentration of the essential oil in the plant.

Essential oils may be classified using different criteria: consistency, origin, chemical

nature of main components.

The essential oil chemical profile is closely related to the extraction procedure employed, and thus,

choice of appropriate extraction method is crucial. On the basis of the properties of the plant material,

the following extraction techniques can be applied: steam distillation, solvent extraction, fractionation

of solvent extracts, maceration, etc.

The techniques commonly applied to assess essential oil physical properties are:

18.2 RELATIVE DENSITY

Relative density is defined as the ratio of the densities of given oil with respect to water at specified

temperature.

Unless otherwise stated, it is based on the ratio of the weight of a liquid in air at 25° to that of an

equal volume of water at the same temperature. Where a temperature is specified in an individual

monograph, the specific gravity is the ratio of the weight of the liquid in air at the specified

temperature to that of an equal volume of water at the same temperature.

18.3 OPTICAL ROTATION

Optical rotation, ‘α’ is the property shown by certain substances of rotating the plane of polarized

light. Such substances are said to be optically active in the sense that they cause incident polarized

light to emerge in a plane forming a measurable angle with a plane of the incident light.


83

The optical rotation of a substance is the angle through which the plane of polarized light is rotated

when polarized light passes through the substance, if liquid, or a solution of the substance. Substances

are described as dextro-rotatory or laevo-rotatory according to whether [α]D25

the plane of polarization

is rotated clockwise or anticlockwise, respectively as determined by viewing towards the light source.

Dextro-rotation is designated (+) and laevo-rotation is designated (-).

The optical rotation unless otherwise specified, is measured at the wavelength of the D line of sodium

(λ= 589.3 nm) at 25°, on a layer 1dm length. It is expressed in degrees.

Optical activity is determined by using a polarimeter, with the angle of rotation depending on a series

of parameters, such as oil nature, the length of column through which the light passes, the applied

wavelength, and the temperature. The degree and direction of rotation are of great importance for

purity assessments, since they are related to the structures and the concentrations of the chiral

molecule in the sample. Each optically active substance has its own specific rotation.

Calculation

Calculate the specific optical rotation using the formulae, dextro-rotation and laevo-rotation being

designated by (+) and (-) respectively.

[α]D20

= α / ld20

Where, α = corrected observed rotation, in degress, at 20°

D = D line of sodium light (l = 589.3nm)

l = length of the polarimeter tube in dm

d20

= specific gravity of the liquid or solution at 20°

18.4 REFRACTIVE INDEX

The refractive index (n) of a substance with reference to air is the ratio of the sin of the angle of

incidence to the sin of the angle of refraction of a beam of light passing from air into the substance. It

varies with the wavelength of the light used in its measurement.

Unless otherwise specified in individual monograph, the refractive index, nD20

is measured at

20°±0.5° with reference to the wavelength of the d-line of sodium (λ=589.3 nm).

NOTE: The temperature should be carefully adjusted and maintained since the refractive index varies

significantly with temperature.

18.5 MONOGRAPH DEVELOPMENT OF ESSENTIAL OIL/ VOLATILE OIL

a) Naming the monograph


84

The family of the plant along with its genus and species along with common name/ popularly

known name is mentioned as the name of the monograph.

b) Source of Essential oil

Essential oils are generally derived from one or more plant parts, such as flowers (e.g. rosemary,

lavender), leaves (e.g. mint, lemongrass), leaves and stems (e.g. Clove), bark (e.g. cinnamon,

cassia, canella), roots (valerian), seeds (e.g, castor, cumin, nutmeg), fruits (coriander, cumin),

rhizomes (e.g. ginger) and gums or oleoresin exudations.

c) Active constituents

The minimum content of quantifiable chief marker constituents shall be provided.

Eg:- Lemon Grass oil is the essential oil obtained by steam distillation of the leaves of

Cymbopogon flexuosus (Fam. Graminae). It contains not less than 70.0 per cent and not more

than 90.0 per cent of citral. Not less than 1.56 per cent and not more than 8.0 per cent of methyl

heptanoate.

d) Category

Therapeutic category and strength is specified wherever possible.

e) Description

This section contains a brief description of the organoleptic characters of the drug. The

information given is not to be regarded as representing mandatory requirements but is important

in preliminary evaluation. The colour of the drug, where this is characteristic.

The odour of the drug, no reference is made to taste of the drug. Eg:- A clear, pale yellow to

amber liquid, free from sediment, suspended matter and characteristic lemon like odour.

f) Identification

This section includes tests performed to identify the drug. It can be performed by GC, TLC and

flash point where applicable. All the identifications mentioned below are not necessarily

included: some may be absent when they are not feasible or are not significant for the purpose of

identification.

g) Gas Chromatography (2.4.13)

Where GC is used in a test or assay, it may also be referred to under Identification. Reference GC

chromatograms are mentioned in Volume I of IP. The chromatograms to be reproduced shall

consist of 2 parts:


85

Part 1: Depicting the running of a gas chromatograph, as per the method given in the monograph

using the Phytochemical Reference Substance(s) (PRS)/ marker compound. Such chromatogram

shall be labeled clearly about X and Y axis, what they depict, clearly point out the “Retention

Time at which the peak of the PRS/ marker compound appears”. The peak shall be labeled

suitability, suffixed by a small arrow pointing to the peak. In such chromatogram, if any internal

standard used or any other compounds whose identity and retention time are known appear, they

may also be labeled.

Part 2: Depicting the running of a GC, as per the method given in the monograph using the

sample of the material under test. Such chromatogram shall be labeled clearly about X and Y

axis, what they depict, clearly point out the Retention Time at which the peak where the marker

compound under analysis is appearing. The peak shall be labeled with the name of the marker

compound, suffixed by a small arrow pointing to the peak. In such chromatogram, if any internal

standard if used or any other compounds whose identity and retention time are known, appear,

they may be also labeled. Other peaks that may appear in the chromatogram, whose chemical

identity is not known, need not be labeled.

On top of all pages where such typical chromatograms are reproduced, following text should be

written.

“Reproduced below is Typical chromatogram of an essential oil for which a monograph

appears in this edition of IP”. Each typical chromatogram bears the name of the monograph below it

Example: GC chromatogram of XYZ Seed Oil”:

h) Flash Point (2.4.44)

The flash point of a volatile material is the lowest temperature at which it can vaporize to form

an ignitable material in air. At the flash point, the vapor may cease to burn when the source of

ignition is removed. The flash point is often used as as a descriptive characteristic of liquid fuel,

Res

pon

se (

mV

)


86

and it also used to help characterize the fire hazards of liquids. “Flash point” refers to both

flammable liquids and combustible liquids.

Flash point as per IP 2014 general chapter 2.4.44 is incorporated in identification.

Other identification test respective to each monograph as applicable are incorporated.

NOTE: THE TEST RESULTS ARE COMPARED WITH THAT OF REFERENCE

i) Tests

All the tests mentioned below are not necessarily included: i.e. may depend upon type of oil

(Essential oil/volatile oil).

Tests such as Relative density (2.4.29), Refractive index (2.4.27), Optical rotation (2.4.22), Acid

value (2.3.23), Light absorption (2.4.7), Peroxide value (2.3.35), Acetyl value (2.3.22), Hydroxyl

value (2.3.27), Saponification value (2.3.37), Iodine value (2.3.28), Foreign fatty substances,

Melting range (2.4.21) etc.

j) Assay

Wherever possible, an assay by chemical method or GC is carried out. Specific constituents of

the essential oil can be quantified by GC. The chromatographic system consisting of the details

of column, column temperature, mobile phase, detector, flow rate, injection volume and split

ratio.

k) Storage

Distilled oils are normally packed in tightly closable glass containers or glass-lined aluminum

container or aluminum containers and are to be stored and transported at temperature below 25°.

Exposure to heat and light are to be prevented recognizing their high flammability, and potential

oxidative properties.


87

19. EXAMPLES OF SOME HERBAL MONOGRAPHS

RAW HERB

Arjuna

Terminalia arjuna Bark

Arjuna consists of dried stem bark of Terminalia

arjuna (Roxb) Wight &Arn (Fam. Combretaceae)

Arjuna contains not less than 0.02 per cent of

arjungenin calculated on the dried basis.

Category.Antihyperlipidaemic, antihypertensive,


Description.A flat or minutely curved thick pieces of

bark with reddish gray colour and astringent taste.

Identification

A. Macroscopic — Stem bark pieces, flat or minutely

curved, with reddish gray external surface and darker

inner surface. Internal surface has longitudinal minute

ridges. Fractures longitudinal.

B. Microscopic— Cork consisting of 6-10 layers of

elongated cells, phloem broad, medullary rays

uniseriate. Calcium oxalate clusters abundant. Few of

the parenchyma cells contain colouring matter.

C. Determine by thin-layer chromatography (2.4.17),


Mobile phase.A mixture of 5 volumes of toluene, 5

volumes of ethyl acetate and 0.5 volume of acetic

acid.

Test solution. Reflux 2 g of coarsely powdered

substance under examination with 50 ml of

chloroform for 15 minutes, cool and filter. Reflux the

residue further with 50 ml of chloroform. Combine

the filtrate and concentrate under vacuum to dryness.

Dissolve the residue in 10 ml of ethanol at 50° for 10

minutes and filter.

Reference solution. Reflux 1 g of arjuna RS with 50

ml of chloroform for 15 minutes, cool and filter.

Reflux the residue further with 50 ml of chloroform.

Combine the filtrate and concentrate under vacuum to

dryness. Dissolve the residue in 5 ml of ethanol at 50°

for 10 minutes and filter.



Dry the plate in air and examine under ultraviolet light

at 254 nm and 365 nm, spray with 10 per cent w/v

solution of sulphuric acid in methanol. Heat the plate

at 110º for 5 minutes and examine in day light. The

chromatographic profile of the test solution is similar

to that of the reference solution.

Tests

Foreign organic matter (2.6.1). Not more than 2.0

per cent.

Ethanol-soluble extractive (2.6.2). Not less than

20.0 per cent.

Water-soluble extractive (2.6.3). Not less than 20

per cent by method I.


Acid-insoluble ash (2.3.19). Not more than 2.0 per

cent.




88



Microbial contamination (2.2.9).Complies with the



Test solution. Reflux 5 g of coarsely powdered

substance under examination with 50 ml of

chloroform for 15 minutes, cool and filter. Reflux the

residue further with 50 ml of chloroform, cool and

filter. Combine the filtrates and concentrate under

vacuum to dryness, then extract dried residue with 10

ml of ethanol at 50° for 10 minutes and filter.

Reference solution. A 0.1 per cent w/v solution of

arjungenin RS in ethanol.


– a stainless steel column 25 cm x 4.6 mm packed

with octadecylsilane bonded to porous silica (10

µm),

– mobile phase: A. acetonitrile (70 per cent) in

water,

B. acetonitrile (30 per cent) in

water,

– a gradient programme using the condition given

below,

– flow rate: 1.2 ml per minute,


– injection volume: 20 µl

Time Mobile phase A Mobile phase B

(in min) (per cent v/v) (per cent v/v)

0 30 70

10 50 50

30 70 30

50 30 70

Inject the reference solution. The test is not valid

unless the relative standard deviation for the replicate

injections for the analyte peak corresponding to

arjungenin is not more than 2.0 per cent.


Calculate the contents of arjungenin.

Storage. Store protected from light, heat, moisture

and against attack by insects and rodents.

EXTRACT

Arjuna Dry Extract

Arjuna Dry Extract is obtained by extracting Arjuna

(Terminalia arjunaWight and Arn,Fam. Combretaceae)

bark with methanol or any other suitable solvent and

evaporation of solvent.

Arjuna Dry Extract contains not less than 90.0 per cent

w/w and not more than 110.0 per cent w/w of stated

amount of the arjunolic acid on the dried basis. It may

contain suitable added substances.

Category. Antihyperlipidaemic, antihypertensive,


Usual strength. 60 per cent w/w.

Description. Alight brown to beige powder with or

without red tinge.

Identification


89



Mobile phase. A mixture of 92 volumes of chloroform

and 8 volumes of methanol.




arjunolic acid RS in the methanol.

Apply to the plate 5 µl of each solution as bands 10 mm

by 2 mm. Allow the mobile phase to rise 8 cm. Dry the

plate in air and spray with anisaldehydesulphuric acid

reagent. Heat the plate at 110° for 10 minutes and

examine the plate under ultraviolet light at 365 nm and

day light. The chromatographic profile of the test

solution is similar to that of the reference solution.

B. Determine by thin-layer chromatography (2.4.17),


Mobile phase.A mixture of 35 volumes of chlorofom,

10 volumes of methanol and 2 volumes of water.




arjugenin RS in the methanol.

Apply to the plate 5 µl of each solution as bands 10 mm

by 2 mm. Allow the mobile phase to rise 8 cm. Dry the

plate in air and spray with vanillinesulphuric acid

reagent. Heat the plate at 110° for 10 minutes and

examine the plate under ultraviolet light at 365 nm and

day light. The chromatographic profile of the test

solution is similar to that of the reference solution.

Tests

Ethanol-soluble extractive (2.6.2). Not less than 80.0

per cent.






Microbial contamination (2.2.9).Complies with the



Test solution. Shake a quantity of the extract under

examination containing about 50 mg of arjunolic acid in

50.0 ml of the methanol, filter.

Reference solution.A 0.1 per cent w/v solution of

arjunolic acidm RS in methanol.


– a stainless steel column 25 cm x 4.6 mm packed

with octadecylsilane bonded to porous silica (5

µm),

– mobile phase: a mixture of 35 volumes of 5

-cyclodextrinand 65 volumes of methanol,

– flow rate: 1 ml per minute,


– injection volume: 20 µl.

Inject the reference solution. The test is not valid unless

the relative standard deviation for replicate injections is

not more than 2.0 per cent.


Calculate the content of the arjunolic acid the extract.

Storage. Store protected from heat and moisture.


90

ESSENTIAL OILS

Basil Oil

Tulsi ka tail

Basil Oil is the essential oil obtained by steam

distillation of part of plant (leaves and flowering tops)

Ocimum basilicum (Fam.Lamiaceae).

Basil Oil contains not less than 50.0 per cent and not

more than 75.0 per cent of methyl chavicol, and not less

than 15.0 per cent and not more than 25.0 per cent of

limonene.

Description.A clear, pale yellow to amber liquid, free

from sediment and suspended matter.

Identification

A. Determine by gas chromatography (2.4.13).

Test solution. A 2.0 per cent w/v solution of the oil

under examination in ethanol (95 per cent).

Reference solution.A 2.0 per cent w/v solution of basil

oil RS in ethanol (95 per cent).

Use chromatographic system described in the Assay.

The peaks in the chromatogram obtained with the test

solution corresponds to the peaks in the chromatogram

obtained with the reference solution.

B. Flash point (2.4.44). Not less than 100.0°.

Tests

Relative density (2.4.29).0.9300 to 0.960.

Refractive index (2.4.27). 1.488 to 1.500.

Optical rotation (2.4.22).– 6.0° to +7.5°.

Acid value (2.3.23). Not more than 1.0.

Assay. Determine by gas chromatography (2.4.13).

Test solution.A 2.0 per cent w/v solution of the oil

under examination in ethanol (95 per cent).

Reference solution(a).A 2.0 per cent w/v solution of

methyl chavicol RS in ethanol (95 per cent).

Reference solution (b).A 2.0 per cent w/v solution of

limonene RS in ethanol (95 per cent).


– a capillary column 30 m x 0.25 mm, coated with

methyl 5 per cent phenylpolysiloxane,

– temperature:

column. 50° for 1 minute, increase from 50° to

220° at a rate of 10° per minute and maintain at

this temperature for 13 minutes

– inlet port at 250º and detector at 280º,

– detection by flame ionization detector.

– flow rate 1 ml per minute using nitrogen as carrier

gas.

– injection volume: 1.0 µl.

– split ratio : 1 : 25

Inject reference solutions (a), (b) and the test solution.

Calculate the content of methyl chavicol and limonene

in the oil using area normalisation procedure.

Storage. Store protect from light and moisture.


91

TINCTURE

Belladonna Tincture

Belladonna Tincture obtained from Belladonna leaf or

roots of one or more of the cultivated varieties of

Atropa belladonna Linn. or A. acuminata Royle ex

Lindley (Fam. Solanaceae) or a mixture of both species.

Belladonna Tincture contains not less than 90 per cent

w/w and not more than 110 per cent w/w of total

alkaloids, calculated as hyoscyamine, C17H23NO3.

Category. Anticholinergic.

Usual strength. 0.03 per cent w/w.

Description. A clear green or brownish green liquid.

Identification



Mobile phase. A mixture of 10 volumes of anhydrous

formic acid, 10 volumes of water, 30 volumes of

methyl ethyl ketone and 50 volumes of ethyl acetate.

Test solution. Evaporate 10 ml of the tincture under

examination in a water-bath at 40° under reduced

pressure. Dissolve the residue in 1.0 ml of the

methanol.

Reference solution. Dissolve 1 mg of chlorogenic acid

RS and 2.5 mg of rutin RS in 10 ml of the methanol.



Dry the plate in air, heat the plate at 110° for 10

minutes. Spray the warm plate with 1 per cent v/v

solution of diphenylboric acid aminoethyl ester in

methanol. Allow to cool and spray with 5 per cent v/v

solution of macrogol400 in methanol, allow the plate

to dry in air for 30 minutes and examine under

ultraviolet light at 365 nm. The chromatographic

profile of the test solution is similar to that of the

reference solution.

B. Atropine. Determine by thin-layer chromatography

(2.4.17), coating the plate with silica gel G.

Mobile phase. A mixture of 3 volumes of strong

ammonia solution, 7 volumes of water and 90 volumes

of acetone.

Test solution. To 15.0 ml of the tincture under

examination, add 15 ml of 0.05M sulphuric acid and

filter. Add 1 ml of strong ammonia solution to the

filtrate, with two quantities, each of 10 ml, of peroxide-

free ether, separate the ether layer by centrifugation if

necessary, dry the combined ether extracts over

anhydrous sodium sulphate, filter and evaporate to

dryness on a water-bath. Dissolve the residue in 0.5 ml

of methanol.

Reference solution. Dissolve 50 mg of hyoscyamine

sulphate in 9 ml of methanol and 15 mg of hyoscine

hydrobromide in 10 ml of methanol, separately. Mix 1.8

ml of the hyoscine hydrobromide solution and 8 ml of

the hyoscyamine sulphate solution.

Apply to the plate 20 µl and 40 µl of each solution as

bands 10 mm by 2 mm. Allow the mobile phase to rise

10 cm. Dry the plate at 105° for 15 minutes, spray with

potassium iodobismuthate solution, dry the plate and

spray with sodium nitrite solution until the plate is

transparent. Examine the plate after 15 minutes in day

light. The chromatogram obtained with the test solution


92

corresponds to the chromatogram obtained with the

reference solution.

Tests

Ethanol (2.3.45). 64 to 69 per cent v/v by method III.

Assay. Evaporate 50.0 g of the tincture under

examination to a volume of about 10 ml. Transfer

quantitatively to a separating funnel, with the minimum

volume of ethanol (70 per cent v/v). Add 5 ml of strong

ammonia solution and 15 ml of water. Extract with

three quantities, each of 40 ml of a mixture of 1 volume

of dichloromethane and 3 volumes of peroxide-free

ether, carefully to avoid emulsion, until the alkaloids

are completely extracted. Combine the dichloromethane

and ether extracts; concentrate the solution to a volume

of about 50 ml by heating on a water-bath. Transfer the

resulting solution quantitatively to a separating funnel,

rinsing with peroxide-free ether. Add a quantity of

peroxide-free ether equal to at least 2.1 times the

volume of the solution to produce a layer having a

density well below that of water. Extract the resulting

solution with minimum of three quantities, each of 20

ml of 0.25M sulphuric acid until the alkaloids are

completely extracted. Separate the layers by

centrifugation if necessary and transfer the layers to a

separating funnel. Make the combined layers alkaline

with strong ammonia solution and extract with

minimum of three quantities, each of 30 ml of

dichloromethane until the alkaloids are completely

extracted. Combine the dichloromethane and ether

extracts, add 4 g of anhydrous sodium sulphate and

allow standing for 30 minutes with occasional shaking.

Decant the dichloromethane and filter. Wash the

sodium sulphate with three quantities, each of 10 ml of

dichloromethane. Combine the dichloromethane and

ether extracts, evaporate to dryness on a water-bath.

Heat the residue in an oven at 105° for 15 minutes.

Dissolve the residue in a few ml of dichloromethane,

evaporate to dryness on a water-bath and heat the

residue in an oven at 105° for 15 minutes again.

Dissolve the residue in a few ml of dichloromethane.

Add 20.0 ml of 0.01M sulphuric acid and remove the

dichloromethane by evaporation on a water-bath.

Titrate the excess of acid with 0.02M sodium hydroxide

using methyl red mixed solution as indicator.

1 ml of 0.01 M sulphuric acid is equivalent to 0.005788

g of total alkaloids calculated as hyoscyamine.

Calculate the content of total alkaloids with reference to

the dried material


93

20. HERBAL DRUGS MANUFACTURERS/SUPPLIERS IN INDIA

A non-exhaustive source for extracts and marker compounds suppliers are given below:

S.No MANUFACTURER/

SUPPLIER

ADDRESS TELEPHONE / FAX /

E-MAIL

1. Asthagiri Herbal Research

Foundation

7/1, Sundaram Colony, Thirumalesai St, Tambaram

East, Tambaram East, Chennai, Tamil Nadu 600059

044 2239 7645

2. Aayurmed Biotech Private

Limited

3, Satyam I-B, Raheja Complex, Nr Times of India

Press, Malad, Mumbai, Maharashtra 400 097,

022-32919302

3. Alchem International Private

Ltd.

201, Empire Plaza, Mehrauli - Gurgaon Road,

Sultanpur, New Delhi-110 030

0129-4266000

0129-2307192

4. Central Institute of Medicinal

and aromatic Plants

Central Institute of Medicinal and Aromatic Plants

P.O. - CIMAP, Near Kukrail Picnic Spot,

Lucknow - 226 015

91-522 - 2718524

5. Chromadex 1005 Muirlands Blvd, Suite G, Irvine, CA 92618 USA +1.949.419.0288

Fax: +1.949.419.0294

6. Cymbio Pharma Private

Limited

Cymbio Pharma Private Limited

No. 23/4, N. H. 7, Venkatala, Yelahanka Bengaluru-

560064

09900014078

7. Dabur India Ltd. Unit I & II Plot No.22, Site-IV Sahibabad ,Ghaziabad 0120 – 4378400

Fax : 0120 - 4376924

8. Exotic Naturals 118, Morya House,Off New Link Rd, Andheri (West),

Mumbai, Maharashtra 400053

022- 26733092

9. Govt. Opium and Alkaloids

Works

Mahuabagh, Ghazipur, Uttar Pradesh 233001 011-26417475

Fax: 011-26440667

[email protected]

10. Herbotech Pharmaceuticals VPO Pandori Waraich, Majitha Road,Amritsar, Punjab

143001

0183- 2221025

11. Indo Nacop Chemicals Ltd

23-579, Vishnukundina Nagar,Vinukonda Post -Guntur

District, Andhra Pradesh

08646-272954/ 273513

08646-272954, 09440968849

[email protected]

[email protected]

12. Inga Pharmaceuticals Inga House, Mahakali Road, Andheri East, Mumbai,

Maharashtra 400093,

022-28202932, 28202933

13. Indian Institute of Integrative

Medicine

Post Bag No. 3, Canal Road, Jammu-180001 0191-2584999, 2585222

14. Ivy Comm Systems 1405, Sector-14, Faridabad, Haryana 121 007, India 0129-4006805

15. Konark Herbal & Health Care 332, Adhyaru Industrial Estate Sun Mill Compound,

Lower Parel, Mumbai, Maharashtra-400 013

022-40914300

16. Kumaon Chemical Products Village Shivlalpur Ramnagar Distt., (Nainital),

Uttarakhand - 244715

05947-251243, 08045132916

Fax : 91-05947-251377

17. Min Chem India 117, Loha Bhawan, 93, P. D. Mello Road, Masjid East ,

Mumbai - 400009 Maharashtra

08049442137

18. M R Group Painth Parao, Ramnagar (Distt.) Nainital,

Uttarakhand- 244715

05947-251423

19. Kancor Ingredients Ltd No.VII/138, Kancor Road, Angamaly South,

Ernakulam, Kerala-683573

0484-3051000, 2456451

20. Kumaon Chemical Products Village Shivlalpur, Ramnagar Distut,

Uttarakhand-244715

05947-251716

21. Natural Remedies Pvt. Ltd.

Plot No. 5B, Veerasandra Industrial Area, 19th K.M.

Stone, Hosur Road, Electronic City (Post), Bangalore,

Karnataka-560 100

080-4020 9999

22. Nature & Nurture Healthcare

Pvt. Ltd.

305, Vardhman City-2, Plaza Commercial Centre, Asaf

Ali Road, New Delhi, Delhi-110002

043524267/ 43752230

23. Naturol BioEnergy Limited Plot No: 1056/1, Road No# 45, Jubilee Hills, 040-23556979, 23541829



94

Hyderabad, Andhra Pradesh-500 033

24. Neeru Enterprises 6, Saleem Manzil, Civil Lines, Rampur, Uttar Pradesh

244 901

0595-2350829/ 2353744

25. Nishant Aromas 424/425, Milan Industrial Estate, Off T J Road, Cotton

Green, Cotton Green, Mumbai, Maharashtra-400033

022- 2471 0058

26. Phyto Concentrate

Phyto Concetrates

Factory Adress 1: Block No - 1145,Near Santej petrol

pump,

Santej, District: Gandhinagar

Factory Adress 2: Plot No 63/1, Industrial Estate,

Express Highway, Halol-389350,

Gujarat, India

02764-286568

02764-286409

[email protected]

27. Pioneer Enterprise

101, Raudat Tahera Street, Mumbai-400003

022-2347 2534

Fax: +9122 2347 0325

[email protected]

[email protected]

28. Plantmed Pharma Pvt. Ltd. No. 1187/4, Kaliamma Temple Road, T. Dasarahalli,

Bangalore, Karnataka- 560057

080-32212625

29. Quad Lifesciences Pvt.Ltd. Village Bhagwas, 6 K.M. Derabassi – Barwala Road,

Derabassi– 140 507 Distt, Mohali, Punjab

30. RYM Exports 23, Anuradha Society, Old Nagardas Rd. Andheri(E),

Mumbai, Maharashtra 400069

022-32956539, 28210025

31. Sami Labs Limited 19/1 & 19/2, I Main, II Phase, Peenya Industrial Area,

Bangalore, Karnataka 560 058,

080-28397973

32. Sanat Products 3rd Floor, Sagar Plaza, Distt Centre, Laxmi Nagar,

Vikas Marg Laxmi Nagar 110092, New Delhi, Delhi

011- 2251 8794

33. Shashi Phytochemical

Industries

1, Old Industrial Area, Alwar, Rajasthan 301001 0144-2373770, 72, 73

34. Sigma Aldrich

DSM-149,150,151, 1st Floor, DLF Towers, Shivaji

Marg, New Delhi-110015

011-6676 2800

Fax: 011-6676

[email protected]

35. Sonic Biochem Extractions 38, Patel Nagar, Indore, Madhya Pradesh 452001 0731-2466456, 2447099

36. Surya Pharmaceutical Ltd. S.C.O: 164-165, Sector 9-C, Chandigarh 160 009 0172-5005000

37. Ultra International 64/1, Site 4, Sahibabad Industrial Area, Ghaziabad,

Uttar Pradesh 201010

0120 438 8500

38. Venbiotech Pvt. Ltd. 10, Chokkanathar Street, Karthikeyan Nagar,

Maduravoyal, Chennai, Tamil Nadu 600095

044-42918171



95

BIBLIOGRAPHY

1. Chandra H, Purohit VK. High Performance Thin Layer Chromatography (HPTLC): A

Modern Analytical Tool for Biological Analysis. Nature and Science 2010;8(10):58-61.

2. Kamboj A. Analytical evaluation of herbal drugs. Drugs Discovery Research in

Pharamocognosy/Analytical Evaluation of Herbal Drugs. (Eds): Vallisuta O, Olimat SM,

2012; 23-29.

3. Mukherjee PK. Quality Control of Herbal Drugs published by Business Horizons 2002; 1:

426-491.

4. WHO’s Quality Control Methods for Medicinal Plant Materials 1998. World Health

Organization, HQ, Geneva.

5. EMEA 1998, Quality of Herbal Medicinal Products Guidelines European Agency evaluation

of Medicinal Products (EMEA), London.

6. Wagna H, Bladt S. Plant Drug Analysis- A Thin Layer Chromatography Atlas from Springer

2009;2: 351-364.

7. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, Nirali Prakashan 31st edition 2005.

8. Meier B, Sprianod. Moden HPTLC a perfect tool for quality control of herbals and their

preparations, Journal of AOAC International 2010; 95(5):1399-1409.

9. Sherma J. Biennial review of planar chromatography 2009-2011. Journal of AOAC

International 2011; 95(4):992-1008

10. Sherma J. Review of HPTLC in drug analysis: 1996-2009. Journal of AOAC International

2010; 93(3):754-764.

11. Sadek PC. Trouble shooting HPLC system: A Bench Manual, Wiley, New York 2000.

12. Wolfender JL. HPLC in natural product analysis: The petechan issue, Planta Medica 2009; 75

(7):719-734.


96

BOOKS FOR FURTHER READING ON HERBS

1. Indian Pharmacopoeia 2014 and Addendum 2015. Ministry of Health and Family Welfare,

Government of India, Indian Pharmacopoeia Commission, Ghaziabad, U P.

2. The Ayurvedic Pharmacopoeia of India 2011. Government of India, Ministry of Health and

Family Welfare, Department of AYUSH, New Delhi. Part I, Volume I to IX.

3. The Ayurvedic Pharmacopoeia of India 2011. Government of India, Ministry of Health and

Family Welfare, Department of AYUSH, New Delhi. Part II, Volume I and II.

4. Ayurvedic Formulary of India1978. Government of India, Ministry of Health and Family

Welfare, Department of AYUSH, New Delhi. Part I and Part II.

5. Pulok K Mukherjee 2009. Peter J Houghton (Editors), Evaluation of Herbal Medicinal

Products, Perspectives on Quality, Safety and Efficacy, Pharmaceutical Press, London.

6. Indian Herbal Pharmacopoeia 2002. Indian Drugs Manufacturers Association, Mumbai, India.

7. Quality Standards of Indian Medicinal Plants 2008. Indian Council of Medical Research,

New Delhi, A K Gupta (Coordinator), Volumes I to XI.

8. Reviews on Indian Medicinal Plants 2011. Indian Council of Medical Research, New Delhi,

A K Gupta and Neeraj Tandon (Editors), Volumes I to VII.

9. Y K Sareen. Illustrated Manual of Herbal Drugs used in Ayurveda 1999. Council of Scientific

and Industrial Research and Indian Council of Medical Research.

10. Major Herbs of Ayurveda 2002. Churchil Livingstone – Elsevier Science, London, Elizabeth

M Williamson.

11. Thin Layer Chromatographic Atlas of Ayurvedic Pharmacopoeial Drugs 2009. Government

of India, Ministry of Health and Family Welfare, Department of AYUSH, New Delhi. Part I,

Volume I and II.

12. Macroscopic and Microscopic Atlas of Pharmacopoeial Drugs 2009. Government of India,

Ministry of Health and Family Welfare, Department of AYUSH, New Delhi. Part I,

Volume I to V.

13. British Herbal Pharmacopoeia 1996. British Herbal Medicine Association, London.

14. British Herbal Compendium Volume I 1992 and Volume II 2006. British Herbal Medicine

Association, London.

15. British Pharmacopoeia 2015. Department of Health Social Services and Public safety,

Medicines and Healthcare products Regulatory Agency (MHRA), London.

16. US Pharmacopoeia National Formulary 2015. United States Pharmacopoeial Convention,

Rockville, MD.

17. The Japanese Pharmacopoeia JP XVI 2011. Ministry of Health, Labour and Welfare.

Pharmaceutical and Medical Device Regulatory Science Society of Japan, Tokyo, Japan


97

18. Pharmacopoeia of the People’s Republic of China. Vol III 2010. Chinese Pharmacopoeia

Commission. Beijing, China.

19. USP Dietary Supplements Compendium 2015 Vol IV. United States Pharmacopoeial

Convention, Rockville, MD.

20. Sukh Dev, Prime Ayurvedic Plant Drugs 2006. Anamaya Publishers (New Delhi) and Anshan

Tunbridge Wells, UK.

21. Malati G Chauhan and APG Pillai. Microscopic Profile of Powdered Drugs used in Indian

Systems of Medicine, Institute of Ayurvedic Medicinal Plant Sciences, Gujarat Ayurveda

University, Jamnagar, India, Volume I to V.


98

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