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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

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Produced and published by

Government of India Ministry of Health and Family welfare 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]

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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.

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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

4

Acknowledgements

6

Abbreviations

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,

25

Processed Herbs and Herbal Products In IP 7.

Standard Operating Procedure for Herbal Extracts Monographs

31

8.

Standard Operating Procedure for Preparation, Qualification, Certification &

35

Supply of Botanicals Reference Substance (BRS) 9.

Standard Operating Procedure for Preparation, Qualification, Certification &

39

Supply of Phytochemicals Reference Substance (PRS) 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

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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 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

Sub-Group on Essential oils Mr. Ramakant Harlal Kha, M/s Nishant Aromatics 424/425, Milan Industrial Estate, Off T J Road, Cotton Green, Mumbai, Maharashtra 400033. Dr. Rahul Singh, M/s Emami (I) Pvt. Ltd. 687 Anandapur, EM Bypass, Kolkatta 700107

Mr. B. Murali (Affiliation - as above)

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 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, Pharmacopoeial Associate

Mr.Ashish Kumar Kushwaha Pharmacopoeial Associate

Dr. Divya Kaushik Pharmacopoeial Associate

Ms. Neha Singh, Pharmacopoeial Associate

Ms. Amandeep Bhatia, Pharmacopoeial Associate

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: 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

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

RAW HERBS

HERBAL EXTRACTS

PROCESSED HERBS/ PHARMACEUTICAL AIDS/ FORMULATIONS

(A)

(B)

(C)

Acacia Ajwain Amalaki Amaltas Amra Anantmula Arjuna Artemisia Ashwagandha Asthisamhrta Bakuci Bala Bassant Belladona Leaf Bhibhitaki Bhringraj Bhuiamla Birmi Brahmi Coleus Daruharidra Roots Daruharidra Stems Draksha Ergot Garcinia Ginseng Gokhru Gudmar Guduchi Guggul resin Haridra Haritaki Hingu Ispaghula Husk

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68.

Ivy Leaf Kalmegh Kaunch Kundru Kutki Lasuna Lavang Lodhra Mandukaparni Manjistha Maricha Methi Mirch Nagakesar Neem Pippali Large Pippali Small Punarnava Sahajana Leaf Sahajana Stick Shankhpushpi Sarpagandha Saunf Senna Leaf Senna Pods Shatavari Shati Sunthi Tulasi Valerian Root Vasaka Vidanga Vijayasara Yasti

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Amla Juice Powder Arjuna Dry Extract Ashwagandha Dry Extract Bassant Dry Extract Belladonna Dry Extract Belladonna Soft Extract Bhibhitaki Aqueous extract Brahmi Extract Coleus Dry Extract Garcinia Aqueous extract Ginseng Dry Extract Gugulipid Haridra Dry extract Haritaki Aqueous extract Haritaki Extract Ivy Leaf Dry Extract Kalmegh Dry Extract Senna Dry Extract Sunthi Extract Tulasi Dry Extract Valerian Dry Extract Vasaka Extarct Yasti Dry Extract Opium Bhuiamla Dry Extract Gudmar Dry Extract Kunduru Dry Extract Mandukaparani Dry Extract Malt Extract

Gross Total: A+ B+ C =

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

Arachis oil Basil Oil Belladonna Tincture Black pepper oil Caraway Oil Cardamom Oil Castor Oil Clove Bud Oil Clove Leaf Oil Clove Stem Oil Coconut Oil Coriander Oil Cumin Oil Dill Seed Oil Prepared Ergot Eucalyptus oil Guar Gum Hydrogenated Castor Oil Lavender Oil Lemon Grass Oil Lemon Oil Lime Oil Mentha Oil Mentha Arvensis Oil Nutmeg Oil Papain Peppermint Oil Rosemary Oil Sarpagandha Powder Sarpagandha Tablets Shellac Starch Thyme Oil Tolu Balsam Tragacanth Opium powder Ipecac Tincture Gugulipid Tablets Senna Tablets

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

Concurrence of Chairman of HPEG for the items for the monograph development based on laid down criteria

Efforts made to obtain 3 samples from different sources

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

Sample authenticated and comparison made with reference substance for monograph development

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

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

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

Examination of public comments

Incorporation of valid comments for adoption in Indian Pharmacopoeia

Herbal monograph published in IP

Feedback from Stakeholders

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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.

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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 17

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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 postharvest 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 UVVisible 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 18

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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.

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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

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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 21

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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 22

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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 23

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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.

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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

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Table 1: Description of number of units of each herbal material Category Woody and available in large pieces –

No. of Units 4-6

Category Stems and roots with smaller

stem, wood, and heartwood, woody roots

diameters

(Eg. Deodar, Erandmool)

(Eg. Ephedra, Manjistha, Kutaki)

Leafy and creepers cut into parts

10 to 12

(Eg. Bhringraj, Neem)

Stigma, Style, Anthers, Small Petals,

No. of Units 8-14

20-40

Buds (Eg.Keshar, Lavang)

Fleshy Dried Rhizomes

4-8

(Eg.Vidarikand, Varahi) Flowers, Larger Petals, Small Fruits

10-20

(Eg.Japakusum, Kusumphool) Bark cut into pieces (Eg. Arjuna, Kutaz)

Minute seeds and parts of seeds (Eg.Vakuchi, Isabgol)

More than 40

Resins, Gums in dried form

4-8

(Eg. Heeng, Babool) 3-8

Minute parts like epidermal hair

5-10

(Eg. Kamela)

Example: Photograph of Arjuna

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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.” 27

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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)

(Under day light after spraying with vanillin sulphuric acid reagent)

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.” 28

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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

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Example: Typical GC chromatogram of Nigella sativa”

p-Cymene

Response (mV)

α- Pinene

“Reproduced below are Typical chromatograms of herbs for which a monograph appear in this edition of IP”.

GC chromatogram of Nigella sativa

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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

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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. 32

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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 Reference solution. A 0.1 per cent w/w solution of Arjuna Dry Extract

arjunolic acid RS in the methanol.

Arjuna Dry Extract is obtained by extracting Arjuna

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

(Terminalia

Arrn,

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

Fam. Combretaceae) bark with methanol or any other

Dry the plate in air and spray with anisaldehyde

suitable solvent and evaporation of solvent.

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

Arjuna Dry Extract contains not less than 90.0 per

minutes and examine the plate under ultraviolet light

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

at 365 nm and day light. The chromatographic profile

stated amount of the arjunolic acid on the dried basis.

of the test solution is similar to that of the reference

It may contain suitable added substances.

solution.

Category. Antihyperlipidaemic, antihypertensive,

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

astringent, cardioprotective, hrdroga.

coating the plate with silica gel GF254.

Usual strength. 60 per cent w/w.

Mobile phase. A mixture of 35 volumes of

Description. A light brown to beige powder with or

chloroform, 10 volumes of methanol and 2 volumes of

without red tinge.

water.

Identification

Test solution. Dissolve 50 mg of the extract under

arjuna

Wight

and

examination with 10.0 ml of methanol and filter. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.

Reference solution. A 0.05 per cent w/w solution of arjugenin RS in the methanol.

Mobile phase. A mixture of 92 volumes of chloroform and 8 volumes of 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.

Test solution. Dissolve 50 mg of the extract under examination with 50.0 ml of methanol and filter.

Dry the plate in air and spray with vanilline sulphuric acid reagent. Heat the plate at 110° for 10 minutes 33

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and examine the plate under ultraviolet light at 365

Chromatographic system

nm and day light. The chromatographic profile of the

– a stainless steel

column 25 cm x 4.6 mm

test solution is similar to that of the reference solution.

packed with octadecylsilane bonded to porous

Tests

silica (5 µm), – mobile phase: a mixture of 35 volumes of 5 mM

Ethanol-soluble extractive (2.6.2).

Not less than

80.0 per cent.

α- cyclodextrin and 65 volumes of methanol, – flow rate: 1 ml per minute,

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

– spectrophotometer set at 205 nm,

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

– injection volume: 20 µl.

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

Inject the reference solution. The test is not valid

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

unless the relative standard deviation for replicate

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

injections is not more than 2.0 per cent.

Microbial contamination (2.2.9). Complies with the

Inject the reference solution and the test solution.

microbial contamination tests.

Calculate the content of the arjunolic acid in the

Assay. Determine by liquid chromatography (2.4.14).

extract.

Test solution. Shake a quantity of the extract under

Storage. Store protected from heat and moisture.

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.

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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 35

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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.

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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 Labelling requirement for candidate Botanical Reference Substance

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) 37

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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 belowe) “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 Storage: Cool and dry place

IPC

======================================= Indian Pharmacopoeia Commission, Ghaziabad

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

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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 39

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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. PHYTOCHEMICAL REFERENCE SUBSTANCE ___mg Name of PRS, 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 Labelling requirement for candidate Phytochemical Reference Substance

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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-

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“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.

PHYTOCHEMICAL REFERENCE SUBSTANCE Name of PRS, IP (as given in IP Monograph): Lot No: BM IP 04062015 Date of Packing: June 15 Storage: Cool and dry place ======================================== Indian Pharmacopoeia Commission, Ghaziabad

9.2.5

IPC

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.

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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.

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IDENTIFICATION OF HERBAL MATERIAL

Monograph available in other Pharmacopoeias Yes Compliance with all identification tests (macroscopic, microscopic and chromatographic method) in

available pharmacopoeial references ant Pharmacopoeia

No 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

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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

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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.

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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 47

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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. 48

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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.

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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.

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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.

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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 “nonphysiological” 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.

Weight of ash (g) Percentage of ash with reference to air dried drug =

X 100 Weight of the drug (g)

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,

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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.

Percentage of acid insoluble ash with reference to air dried drug =

Weight of acid insoluble ash X 100 Weight of the drug

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 soluble ash X 100 Weight of the drug

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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 Percentage of ethanol soluble extractive =

X 100 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 IProceed 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 IIAdd 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. 54

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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:

Percentage of water soluble extractive =

Weight of residue Weight of the drug

X 100

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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 wellmixed 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.

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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.

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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

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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.

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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)

Rƒ =

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

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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

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SOLVENT SYSTEMS

SOLVENT RATIO

SORBENTS

CHEMICAL CONSTITUENT

Essential Oils Toluene: Ethyl acetate Toluene

93:7

Silica gel 60F254

Anethole/ methylchavicol or safrole, eugenol

-

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

Silica gel 60F254

Suitable for major alkaloids of most drugs

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

40:40:6:2

Silica gel 60F254

n-Heptane: Ethylmethyl ketone: Methanol

58:34:8

Silica gel 60F254

Cyclohexane: Chloroform : Glacial acetic acid

45:45:10

Silica gel 60F254

Ethyl acetate: Methanol:Water

Toluene: Acetone: Ethanol : conc. Ammonia

70:20:10

Opium alkaloids Rauwolfia alkaloids Berberine and protoberberine type alkaloids

Bitter Drugs Ethyl acetate: Methanol: Water

77:13.5:8

Silica gel 60F254

General system for screening

70:30:2

Silica gel 60F254

Amarogentin

95:5

Silica gel 60F254

Quassin

Iso-octane: Isopropanol: Formic acid

83.5:16.5:0.5

Silica gel 60F254

n- heptanes: Isopropanol: Formic acid

90:15:0.5

Silica gel 60F254

95:5

Silica gel 60F254

Acetone: Choloroform: Water Chloroform: Methanol

Chloroform: Ethanol

Humulone and lupulone Bitter acids Cucurbitacins 62

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Anthracene derivatives Ethyl acetate: Methanol: Water

100:13.5:10

Silica gel 60F254

n-propanol: Ethyl acetate: Water: Glacial acetic acid

40:40:29:1

Silica gel 60F254

Light petroleum: Ethyl acetate :Formic acid

75:25:1

Silica gel 60F254

Toluene: Ethylformate: Formic acid

50:40:10

Silica gel 60F254

All anthracene drug extracts Senna Anthraquinone aglycones Hypericin

Cardiac Glycosides 100:13.5:10 Ethyl acetate: Methanol: Water

Or

Silica gel 60F254

Applicable system for cardiac glycosides

81:11:8 Ethyl acetate: Methanol: 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

Ethyl acetate: Formic acid: 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

75:16.5:8.5

Silica gel 60F254

Flavonoids

50:40:10

Silica gel 60F254

Flavonoid aglycones

Chloroform: Acetone: Formic acid Toluene: Ethylformate: Formic acid

Coumarins Toluene: Ether

(1:1) saturated With 10% acetic

Silica gel 60F254

Universally accepted for coumarin aglycones

Silica gel 60F254

Used for higher Rf

Acid Ethyl acetate

-

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Ethyl acetate: Formic acid: Glacial acetic acid: Water

100:11:11:27

Silica gel 60F254

For polar coumarins

Silica gel 60F254

Podophyllins

Lignans Chloroform: Methanol (6cm) followed by toluene-acetone

90:10 followed by 65:35 Pungent principles from plants

Toluene: Ethylacetate Toluene: Ethyl ether: Dioxan Diethyl ether (saturated chamber)

70:30

Silica gel 60F254

Piper

62.5:21.5:16

Silica gel 60F254

General for pungent principles

-

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)

Source: In Mukherjee PK (2002). Thin Layer Chromatography. Quality Control of Herbal Drugs. Business Horizons publisher. 1 st 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

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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.

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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.

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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. 1 2 3

4

5 6 7 8 9 10

FEATURES Technique Layer Sample application Solid support PC connectivity Method storage Validation Quantitative analysis Sample holder Wavelength range

TLC Manual Lab made/ Precoated

HPTLC Semiautomatic Precoated

Circular

Rectangular or Circular

Silica gel , Alumina & Kieselguhr etc..

Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes etc

No

Yes

No

Yes

No

Yes

Difficult

Relatively easier

Capillary/ Pipette

Syringe

254 or 366 nm, visible

190 to 800nm, monochromatic

11

Analysis Time

Slower

12

Scanning

Not possible

13

Accuracy

Low

Shorter migration distance and the analysis time is greatly reduced Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer 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 co67

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elute with other substances. Procedure for HPTLC method development is outlined as follows (Figure1) :

FIGURE 1 Schematic procedure for HPTLC method development

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

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 reversedphase 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. 68

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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 69

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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

Trachyspermum ammi (Ajwain)

Antiasthmatic, carminative, germicidal, antioxidant

Toluene : ethyl acetate [93:7]

Anisaldehyde sulphuric acid reagent

2

Emblic myrobalan (Amalaki)

Toluene : ethyl acetate Antiscorbutic, antacid, : glacial acetic acid: carminative, formic acid hepatoprotective [20:45:20:5]

Anisaldehyde sulphuric acid reagent

3

Cassia fistula (Amaltas)

Purgative, diuretic, antipruritic, febrifuge

1.

Toluene : ethyl acetate : formic acid : methanol [3:3:0.8:0.2]

-

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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

Toluene : ethyl acetate : 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 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.

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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 72

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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.

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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.

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Normal phase(adsorpt ion)

Unmodified silica packing

Normal phase(bonded )

CN, NH2 bonded phases

Hexane Soluble

Organic Soluble

MeOH, MeOH/H2O Soluble Reverse phase bonded

MW2000

Sample

Organic Soluble

Small molecule GPC

Hydrophobic small pore packing’s

Non-ionic

Reverse phase(bonded)

C18, C8, phenyl, C4 , CN bonded phases

Ionic (including metal chelates and transition metals)

Reverse phase (with ionizationcontrol)

C18, C8, phenyl, C4 , CN bonded phases (with pH control)

Reverse phase (paired ion)

C18, C8, phenyl, C4 , CN bonded phases (with pH control)

Ion exchange

Anion and cation exchange columns

Chiral chromatography

Chiral chromatography

Chiral chromatography

Water Soluble

C18, C8, phenyl, C4 , CN bonded phases

Chiral phases (eg. Pirkle columns)

Chiral phases (eg. Pirkle columns)

Chiral phases (eg. Pirkle columns)

Reverse phase (with ionizationcontrol)

C18, C8, phenyl, C4 , CN bonded phases (with pH control)

Reverse phase (paired ion)

C18, C8, phenyl, C4 , CN bonded phases (with pH control)

Ion exchange

Anion and cation exchange columns

Fig 1. Choice of HPLC method

17.3 HPLC COLUMN

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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 : S.No. 1 2 3 4 5 6 7 8 9 10 11 12

Properties of solvents commonly used in HPLC.

Solvent Hexane Carbon tetrachloride Chloroform Methylene chloride Tetrahydrofuran Diethyl ether Acetone Ethyl acetate Acetonitrile Isopropanol Methanol Water

Polarity

UV cut off

Nonpolar Nonpolar

Miscible with water No No

Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Nonpolar Polar

No No Yes No Yes Poorly Yes Yes Yes Yes

245 235 215 215 330 260 190 210 205 -

200 263

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 normalphase 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

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 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.

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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). 78

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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

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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

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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.

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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. 82

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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 20

d = 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 83

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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: 84

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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

Response (mV)

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, 85

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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.

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19. EXAMPLES OF SOME HERBAL MONOGRAPHS RAW HERB Dissolve the residue in 10 ml of ethanol at 50° for 10 Arjuna

minutes and filter.

Terminalia arjuna Bark Arjuna consists of dried stem bark of Terminalia arjuna (Roxb) Wight &Arn (Fam. Combretaceae)

Reference solution. Reflux 1 g of arjuna RS with 50

Arjuna contains not less than 0.02 per cent of

ml of chloroform for 15 minutes, cool and filter.

arjungenin calculated on the dried basis.

Reflux the residue further with 50 ml of chloroform.

Category.Antihyperlipidaemic,

Combine the filtrate and concentrate under vacuum to

antihypertensive,

astringent, cardioprotective, hrdroga.

dryness. Dissolve the residue in 5 ml of ethanol at 50°

Description.A flat or minutely curved thick pieces of

for 10 minutes and filter.

bark with reddish gray colour and astringent taste.

Apply to the plate 10 µl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm.

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.

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

B. Microscopic— Cork consisting of 6-10 layers of elongated cells, phloem broad, medullary rays

to that of the reference solution. Tests

uniseriate. Calcium oxalate clusters abundant. Few of Foreign organic matter (2.6.1). Not more than 2.0

the parenchyma cells contain colouring matter. C. Determine by thin-layer chromatography (2.4.17),

per cent. Ethanol-soluble extractive (2.6.2).

coating the plate with silica gel GF254. Mobile phase.A mixture of 5 volumes of toluene, 5

Not less than

20.0 per cent.

volumes of ethyl acetate and 0.5 volume of acetic

Water-soluble extractive (2.6.3). Not less than 20

acid.

per cent by method I.

Test solution. Reflux 2 g of coarsely powdered

Total ash (2.3.19). Not more than 30.0 per cent.

substance

Acid-insoluble ash (2.3.19). Not more than 2.0 per

under

examination

with

50

ml

of

chloroform for 15 minutes, cool and filter. Reflux the

cent.

residue further with 50 ml of chloroform. Combine

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

the filtrate and concentrate under vacuum to dryness.

test for heavy metals, Method B (20 ppm). 87

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Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 5 g by drying in an oven at 105°.

– a gradient programme using the condition given below,

Microbial contamination (2.2.9).Complies with the

– flow rate: 1.2 ml per minute,

microbial contamination tests.

– spectrophotometer set at 210 nm,

Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 5 g of coarsely powdered substance

under

examination

with

50

ml

of

– injection volume: 20 µl Time

Mobile phase A Mobile phase B

(in min) (per cent v/v)

(per cent v/v)

0

30

70

residue further with 50 ml of chloroform, cool and

10

50

50

filter. Combine the filtrates and concentrate under

30

70

30

vacuum to dryness, then extract dried residue with 10

50

30

70

chloroform for 15 minutes, cool and filter. Reflux the

ml of ethanol at 50° for 10 minutes and filter.

Inject the reference solution. The test is not valid

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

unless the relative standard deviation for the replicate

arjungenin RS in ethanol.

injections for the analyte peak corresponding to

Chromatographic system

arjungenin is not more than 2.0 per cent.

– a stainless steel column 25 cm x 4.6 mm packed

Inject the reference solution and the test solution.

with octadecylsilane bonded to porous silica (10

Calculate the contents of arjungenin.

µm),

Storage. Store protected from light, heat, moisture

– mobile phase: A. acetonitrile (70 per cent) in

and against attack by insects and rodents.

water, B. acetonitrile (30 per cent) in water,

EXTRACT Category. Arjuna Dry Extract Arjuna Dry Extract is obtained by extracting Arjuna

Antihyperlipidaemic,

antihypertensive,

astringent, cardioprotective, hrdroga. Usual strength. 60 per cent w/w.

(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

Description. Alight brown to beige powder with or

amount of the arjunolic acid on the dried basis. It may

without red tinge.

contain suitable added substances.

Identification

88

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

Ethanol-soluble extractive (2.6.2). Not less than 80.0

coating the plate with silica gel GF254.

per cent.

Mobile phase. A mixture of 92 volumes of chloroform

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

and 8 volumes of methanol.

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

Test solution. Dissolve 50 mg of the extract under

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

examination with 50.0 ml of methanol and filter.

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

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

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

arjunolic acid RS in the methanol.

Microbial contamination (2.2.9).Complies with the

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

microbial contamination tests.

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

Assay. Determine by liquid chromatography (2.4.14).

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

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.

day light. The chromatographic profile of the test solution is similar to that of the reference solution.

Reference solution.A 0.1 per cent w/v solution of arjunolic acidm RS in methanol.

B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase.A mixture of 35 volumes of chlorofom, 10 volumes of methanol and 2 volumes of water. Test solution. Dissolve 50 mg of the extract under examination with 10.0 ml of methanol and filter.

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

-cyclodextrinand 65 volumes of methanol,

Reference solution. A 0.05 per cent w/w solution of

– flow rate: 1 ml per minute,

arjugenin RS in the methanol.

– spectrophotometer set at 205 nm,

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

– injection volume: 20 µl.

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

Inject the reference solution. The test is not valid unless

plate in air and spray with vanillinesulphuric acid

the relative standard deviation for replicate injections is

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

not more than 2.0 per cent.

examine the plate under ultraviolet light at 365 nm and

Inject the reference solution and the test solution.

day light. The chromatographic profile of the test

Calculate the content of the arjunolic acid the extract.

solution is similar to that of the reference solution. Storage. Store protected from heat and moisture. Tests

89

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ESSENTIAL OILS Basil Oil Tulsi ka tail

Test solution.A 2.0 per cent w/v solution of the oil under examination in ethanol (95 per cent).

Basil Oil is the essential oil obtained by steam distillation of part of plant (leaves and flowering tops) Ocimum basilicum (Fam.Lamiaceae).

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). Chromatographic system

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

– a capillary column 30 m x 0.25 mm, coated with methyl 5 per cent phenylpolysiloxane, – temperature:

limonene.

column. 50° for 1 minute, increase from 50° to

Description.A clear, pale yellow to amber liquid, free

220° at a rate of 10° per minute and maintain at

from sediment and suspended matter.

this temperature for 13 minutes

Identification

– inlet port at 250º and detector at 280º, – detection by flame ionization detector.

A. Determine by gas chromatography (2.4.13). Test solution. A 2.0 per cent w/v solution of the oil

– flow rate 1 ml per minute using nitrogen as carrier gas.

under examination in ethanol (95 per cent).

– injection volume: 1.0 µl.

Reference solution.A 2.0 per cent w/v solution of basil

– split ratio : 1 : 25

oil RS in ethanol (95 per cent).

Inject reference solutions (a), (b) and the test solution.

Use chromatographic system described in the Assay.

Calculate the content of methyl chavicol and limonene

The peaks in the chromatogram obtained with the test

in the oil using area normalisation procedure.

solution corresponds to the peaks in the chromatogram

Storage. Store protect from light and moisture.

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). 90

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TINCTURE to dry in air for 30 minutes Belladonna Tincture

and

examine under

ultraviolet light at 365 nm. The chromatographic

Belladonna Tincture obtained from Belladonna leaf or roots of one or more of the cultivated varieties of

profile of the test solution is similar to that of the

Atropa belladonna Linn. or A. acuminata Royle ex

reference solution.

Lindley (Fam. Solanaceae) or a mixture of both species.

B. Atropine. Determine by thin-layer chromatography

Belladonna Tincture contains not less than 90 per cent

(2.4.17), coating the plate with silica gel G.

w/w and not more than 110 per cent w/w of total

Mobile phase. A mixture of 3 volumes of strong

alkaloids, calculated as hyoscyamine, C17H23NO3.

ammonia solution, 7 volumes of water and 90 volumes

Category. Anticholinergic.

of acetone.

Usual strength. 0.03 per cent w/w.

Test solution. To 15.0 ml of the tincture under

Description. A clear green or brownish green liquid.

examination, add 15 ml of 0.05M sulphuric acid and filter. Add 1 ml of strong ammonia solution to the

Identification

filtrate, with two quantities, each of 10 ml, of peroxide-

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

free ether, separate the ether layer by centrifugation if

coating the plate with silica gel GF254.

necessary, dry the combined ether extracts over

Mobile phase. A mixture of 10 volumes of anhydrous

anhydrous sodium sulphate, filter and evaporate to

formic acid, 10 volumes of water, 30 volumes of

dryness on a water-bath. Dissolve the residue in 0.5 ml

methyl ethyl ketone and 50 volumes of ethyl acetate.

of methanol.

Test solution. Evaporate 10 ml of the tincture under

Reference solution. Dissolve 50 mg of hyoscyamine

examination in a water-bath at 40° under reduced

sulphate in 9 ml of methanol and 15 mg of hyoscine

pressure. Dissolve the residue in 1.0 ml of the

hydrobromide in 10 ml of methanol, separately. Mix 1.8

methanol.

ml of the hyoscine hydrobromide solution and 8 ml of

Reference solution. Dissolve 1 mg of chlorogenic acid

the hyoscyamine sulphate solution.

RS and 2.5 mg of rutin RS in 10 ml of the methanol.

Apply to the plate 20 µl and 40 µl of each solution as

Apply to the plate 40 µl of each solution as bands 10

bands 10 mm by 2 mm. Allow the mobile phase to rise

mm by 2 mm. Allow the mobile phase to rise 15 cm.

10 cm. Dry the plate at 105° for 15 minutes, spray with

Dry the plate in air, heat the plate at 110° for 10

potassium iodobismuthate solution, dry the plate and

minutes. Spray the warm plate with 1 per cent v/v

spray with sodium nitrite solution until the plate is

solution of diphenylboric acid aminoethyl ester in

transparent. Examine the plate after 15 minutes in day

methanol. Allow to cool and spray with 5 per cent v/v

light. The chromatogram obtained with the test solution

solution of macrogol400 in methanol, allow the plate 91

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corresponds to the chromatogram obtained with the

separating funnel. Make the combined layers alkaline

reference solution.

with strong ammonia solution and extract with minimum of three quantities, each of 30 ml of

Tests

dichloromethane until the alkaloids are completely Ethanol (2.3.45). 64 to 69 per cent v/v by method III.

extracted.

Assay. Evaporate 50.0 g of the tincture under

extracts, add 4 g of anhydrous sodium sulphate and

examination to a volume of about 10 ml. Transfer

allow standing for 30 minutes with occasional shaking.

quantitatively to a separating funnel, with the minimum

Decant the dichloromethane and filter. Wash the

volume of ethanol (70 per cent v/v). Add 5 ml of strong

sodium sulphate with three quantities, each of 10 ml of

ammonia solution and 15 ml of water. Extract with

dichloromethane. Combine the dichloromethane and

three quantities, each of 40 ml of a mixture of 1 volume

ether extracts, evaporate to dryness on a water-bath.

of dichloromethane and 3 volumes of peroxide-free

Heat the residue in an oven at 105° for 15 minutes.

ether, carefully to avoid emulsion, until the alkaloids

Dissolve the residue in a few ml of dichloromethane,

are completely extracted. Combine the dichloromethane

evaporate to dryness on a water-bath and heat the

and ether extracts; concentrate the solution to a volume

residue in an oven at 105° for 15 minutes again.

of about 50 ml by heating on a water-bath. Transfer the

Dissolve the residue in a few ml of dichloromethane.

resulting solution quantitatively to a separating funnel,

Add 20.0 ml of 0.01M sulphuric acid and remove the

rinsing with peroxide-free ether. Add a quantity of

dichloromethane by evaporation on a water-bath.

peroxide-free ether equal to at least 2.1 times the

Titrate the excess of acid with 0.02M sodium hydroxide

volume of the solution to produce a layer having a

using methyl red mixed solution as indicator.

density well below that of water. Extract the resulting

1 ml of 0.01 M sulphuric acid is equivalent to 0.005788

solution with minimum of three quantities, each of 20

g of total alkaloids calculated as hyoscyamine.

ml of 0.25M sulphuric acid until the alkaloids are completely

extracted.

Separate

the

layers

by

Combine the dichloromethane and ether

Calculate the content of total alkaloids with reference to the dried material

centrifugation if necessary and transfer the layers to a

92

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20. HERBAL DRUGS MANUFACTURERS/SUPPLIERS IN INDIA A non-exhaustive source for extracts and marker compounds suppliers are given below: S.No

1. 2. 3.

MANUFACTURER/ SUPPLIER Asthagiri Herbal Research Foundation

7/1, Sundaram Colony, Thirumalesai St, Tambaram East, Tambaram East, Chennai, Tamil Nadu 600059

Aayurmed Biotech Private Limited Alchem International Private Ltd.

3, Satyam I-B, Raheja Complex, Nr Times of India Press, Malad, Mumbai, Maharashtra 400 097, 201, Empire Plaza, Mehrauli - Gurgaon Road, Sultanpur, New Delhi-110 030 Central Institute of Medicinal and Aromatic Plants P.O. - CIMAP, Near Kukrail Picnic Spot, Lucknow - 226 015 1005 Muirlands Blvd, Suite G, Irvine, CA 92618 USA

4.

Central Institute of Medicinal and aromatic Plants

5.

Chromadex

6.

Cymbio Pharma Private Limited

7.

Dabur India Ltd.

8.

Exotic Naturals

9.

Govt. Opium and Alkaloids Works

10.

Herbotech Pharmaceuticals

11.

Indo Nacop Chemicals Ltd

12.

Inga Pharmaceuticals

14.

Indian Institute of Integrative Medicine Ivy Comm Systems

15.

Konark Herbal & Health Care

16.

Kumaon Chemical Products

17.

Min Chem India

18.

M R Group

19.

Kancor Ingredients Ltd

20.

Kumaon Chemical Products

21.

Natural Remedies Pvt. Ltd.

13.

22. 23.

ADDRESS

Nature & Nurture Healthcare Pvt. Ltd. Naturol BioEnergy Limited

Cymbio Pharma Private Limited No. 23/4, N. H. 7, Venkatala, Yelahanka Bengaluru560064 Unit I & II Plot No.22, Site-IV Sahibabad ,Ghaziabad 118, Morya House,Off New Link Rd, Andheri (West), Mumbai, Maharashtra 400053 Mahuabagh, Ghazipur, Uttar Pradesh 233001

VPO Pandori Waraich, Majitha Road,Amritsar, Punjab 143001 23-579, Vishnukundina Nagar,Vinukonda Post -Guntur District, Andhra Pradesh

Inga House, Mahakali Road, Andheri East, Mumbai, Maharashtra 400093, Post Bag No. 3, Canal Road, Jammu-180001 1405, Sector-14, Faridabad, Haryana 121 007, India 332, Adhyaru Industrial Estate Sun Mill Compound, Lower Parel, Mumbai, Maharashtra-400 013 Village Shivlalpur Ramnagar Distt., (Nainital), Uttarakhand - 244715 117, Loha Bhawan, 93, P. D. Mello Road, Masjid East , Mumbai - 400009 Maharashtra Painth Parao, Ramnagar (Distt.) Nainital, Uttarakhand- 244715 No.VII/138, Kancor Road, Angamaly South, Ernakulam, Kerala-683573 Village Shivlalpur, Ramnagar Distut, Uttarakhand-244715 Plot No. 5B, Veerasandra Industrial Area, 19th K.M. Stone, Hosur Road, Electronic City (Post), Bangalore, Karnataka-560 100 305, Vardhman City-2, Plaza Commercial Centre, Asaf Ali Road, New Delhi, Delhi-110002 Plot No: 1056/1, Road No# 45, Jubilee Hills,

TELEPHONE / FAX / E-MAIL 044 2239 7645 022-32919302 0129-4266000 0129-2307192 91-522 - 2718524

+1.949.419.0288 Fax: +1.949.419.0294 09900014078

0120 – 4378400 Fax : 0120 - 4376924 022- 26733092 011-26417475 Fax: 011-26440667 [email protected] 0183- 2221025 08646-272954/ 273513 08646-272954, 09440968849 [email protected] [email protected] 022-28202932, 28202933 0191-2584999, 2585222 0129-4006805 022-40914300 05947-251243, 08045132916 Fax : 91-05947-251377 08049442137 05947-251423 0484-3051000, 2456451 05947-251716 080-4020 9999

043524267/ 43752230 040-23556979, 23541829

93

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24.

Neeru Enterprises

25.

Nishant Aromas

26.

Phyto Concentrate

27.

Pioneer Enterprise

28.

Plantmed Pharma Pvt. Ltd.

29.

Quad Lifesciences Pvt.Ltd.

30.

RYM Exports

31.

Sami Labs Limited

32.

Sanat Products

33.

Shashi Phytochemical Industries

34.

Sigma Aldrich

35. 36.

Sonic Biochem Extractions Surya Pharmaceutical Ltd.

37.

Ultra International

38.

Venbiotech Pvt. Ltd.

Hyderabad, Andhra Pradesh-500 033 6, Saleem Manzil, Civil Lines, Rampur, Uttar Pradesh 244 901 424/425, Milan Industrial Estate, Off T J Road, Cotton Green, Cotton Green, Mumbai, Maharashtra-400033 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 101, Raudat Tahera Street, Mumbai-400003

No. 1187/4, Kaliamma Temple Road, T. Dasarahalli, Bangalore, Karnataka- 560057 Village Bhagwas, 6 K.M. Derabassi – Barwala Road, Derabassi– 140 507 Distt, Mohali, Punjab 23, Anuradha Society, Old Nagardas Rd. Andheri(E), Mumbai, Maharashtra 400069 19/1 & 19/2, I Main, II Phase, Peenya Industrial Area, Bangalore, Karnataka 560 058, 3rd Floor, Sagar Plaza, Distt Centre, Laxmi Nagar, Vikas Marg Laxmi Nagar 110092, New Delhi, Delhi 1, Old Industrial Area, Alwar, Rajasthan 301001 DSM-149,150,151, 1st Floor, DLF Towers, Shivaji Marg, New Delhi-110015 38, Patel Nagar, Indore, Madhya Pradesh 452001 S.C.O: 164-165, Sector 9-C, Chandigarh 160 009 64/1, Site 4, Sahibabad Industrial Area, Ghaziabad, Uttar Pradesh 201010 10, Chokkanathar Street, Karthikeyan Nagar, Maduravoyal, Chennai, Tamil Nadu 600095

0595-2350829/ 2353744 022- 2471 0058 02764-286568 02764-286409 [email protected]

022-2347 2534 Fax: +9122 2347 0325 [email protected] [email protected] 080-32212625

022-32956539, 28210025 080-28397973 011- 2251 8794 0144-2373770, 72, 73 011-6676 2800 Fax: 011-6676 [email protected] 0731-2466456, 2447099 0172-5005000 0120 438 8500 044-42918171

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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.

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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

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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.

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e. f.

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