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I N T R 0 D U C T I ~~ Traditional system of medicines i.e. Ayurveda etc., which formed the basis of

health care throughout the world since the earliest days of mankind are still

widely used, and have considerable importance in international trade. India is

sitting on a gold mine of well-recorded and well practiced knowledge of

traditional medicine. But, unlike China, India has not been able to capitalize this

wealth by promoting its use in the developed world despite their renewed

interest in herbal medicines. There are several reasons for this; one of the major

reasons is that our products are not backed by rigorous scientific studies to

establish their safety, efficacy and standards. The demand of ayurvedic medicine

is gearing up at a rapid rate and the scientific community worrying about the

quality, safety and efficacy of the traditional formulation (Bodekar, 2002).

With the widespread use of traditional medicine as well as complementary/

alternative medicine and the rapid expansion of international herbal medicine

markets, the development of national policies and regulations on traditional

medicine has become an important concern for both health authorities and the

public. National policies and regulations on traditional medicine could ensure the

safety, quality and efficacy of herbal therapies and products, and function as

important steps towards integrative health-care systems (WHO, 2001)

In order to promote global acceptance of traditional formulations, there is an

urgent need to evaluate the therapeutic potential of the drugs as per regulatory

guidelines. Ironically, not all of the herbal products are available in standardized

form, which is the minimum requirement for introducing a product in the global

market (WIIO, 2001).

Demand for high quality, safe, effective, and pure natural formulations with

various substances have been growing significantly in the industrial world. In the

J ·a/idation f'!j Preparation Pmau and rinr:rrrrintim: of \om('. tyun<·dit· rormulatiom

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I h b d . I ·1 b . I b ~j past tme, er s an essenha 01 eanng p ants were largely arvested from the

wild and brought to the market without many questions asked about their origin,

method of cultivation, botanical identity, purity, safety, and efficacy. With

further improvement in communication and education, there has been a growing

consciousness in industrialized countries about personal health, environmental

safety, sustainable harvesting, and loss of genetic diversity (Herbal monographs,

1997).

Traditional formulations are medicinal preparations, containing a single plant or

a mixture of two or more different types of medicinal plants. For traditional

formulations, there is a need of scientific proof and clinical validation with

chemical standardization, biological assays, animal models and clinical trials.

Quality assurance of traditional formulations is the prerequisite of credible

clinical trials. According to the draft guidelines stated by the United States Food

and Drug Administration and the European Agency for the evaluation of

medicinal products, various aspects of analysis must be performed for the

purpose of certification of traditional formulations. Currently, there is a

common practice among natural products analysis to select one or more

compounds as either active or markers for purposes of identification and quality

assessment. This problem is compounded when one substance that contains a

specific class of compounds is combined with others containing the same or

different classes of compounds. It is necessary to develop a type of quality

assessment system that adequately meets the complex characteristics of herbals.

Fingerprint analysis by which multiple compounds in single herbal drugs and

finished formulation can be identified represents a rational approach for the

quality assessment of traditional fommlations (Martindale, 1999).

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P f ' I . 'fi I . ffi . d ~1· f ~ rocess o preparatiOn p ays a s1gm !Cant ro e m e ectiveness an qua 1ty o

traditional/ ayurvedic formulation. Process validation is "A documented program

which provides a high degree of assurance that a specific process will

consistently produce a product meeting its predetermined specification and

quality attributes". The quality of pharmaceuticals has been a concern of the

World Health Organization (WHO) since its inception. Many international

authorities and agencies like European agencies for the evaluation of medicinal

product, European Scientific Co-operation of phyto-medicine, U.S. Agencies for

Health Care Policy and Research, European Pharmacopoeia Commission,

Department of Indian System of Medicine have been started creating a new

mechanism to induce a quality control and standardization of botanical and

ayurvedic medicine. It is absolutely necessary to develop reliable analytical

methods in order to control the quality of herbal medicines. Fingerprint

technique was introduced and accepted by WHO, as a powerful tool for

identification and quality evaluation of multi-component herbal/traditional

medicines (WHO Guidelines 1991)

The system of ayurveda, although at present mainly confined to India, but it has

had a much wider reorganization and having prevalence from the past since the

dawn of human civilization. This system has undergone many vicissitudes in the

course of its long and chequered history. However, it still remains the mainstay

of medical relief among the majority of the people in this country. Even in the

neighbouring countries the system of ayurveda is reported to be fairly prevalent

(The Ayurvedic formulary of India, 2003).

In ancient time when the ayurvcdic medicines were developed, preparation,

standardization and quality control of ayurvcdic fommlation maintained by fully

J ·a/idatian (1! Prrrnration rnlct"H and rim:crprintinr: nf \(Jnlt' Ayunt•dic rrmnulatiom

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~! ----~~----~~~~~~--~~------~----~~--~~--~ IN committed professionals. Either failing compliance of complete ayurvedJC

process or adopting short-cuts can result into compromised therapeutic gains in

the present scenario (Patwardhan, 2000, Jain et.al, 2007).

On account of process evolution, commercialization and environment pollution

quality of the ayurvedic formulation declined and they are not as effective as

prehistoric formulation (Mukharjee, 2002).

The traditional ayurvedic text claims that ayurvedic formulation prepared

according to the method given in ayurvedic text will always be superior in

quality, safety, and efficacy (The Ayurvedic formulary of India, 2003,

Bhaisajyaratnawali, 1961 ).

· Ayurvedic formulary of India states that, if the raw materials to be used in a

medicine and stage by stage processes of preparation are standardized, the final

formulation or compound formulation can be expected to confirm the uniform

standards (The Ayurvedic formulary ofindia, 2003).

The proposed work hence is an effort to standardize/ translate the method as

mentioned in the Ayurvedic texts and to develop certain parameters which can

be used as fingerprinting parameter for validation of preparation process and

standardization of ayurvedic formulation.

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----~~ 1.1 Market Potential of traditional! herbal medicine

In developed countries, a resurgence of interest in traditional medicines has

resulted from the preference of many consumers towards products of natural

origin. Recognition of their clinical, pharmaceutical and economic value is still

growing, although this varies widely between cotmtries .The percentage of the

population, which has used CAM at least once, is 48% in Australia, 70% in

Canada, 42% in USA, 38% in Belgium and 90% in Germany. Studies show that

in Africa up to 80% people use traditional medicines, in China TM accounts for

around 40%, and in India 48% of the population having faith in traditional

system of medicine particularly in ayurvedic system of medicine (WHO, 2002-

2005).

It has been postulated that by 2010 at least two-thirds of the United States

population will be using one or more of the alternative therapeutic approaches.

Use of indigenous drugs of natural origin forms a major part of such therapies;

more than 1500 herbals are sold as dietary supplements or ethnic traditional

medicines (WHO, 2001).

Total Indian market for the ayurvedic formulation is of 1000 million dollars a

year while the exports of ayurvedic medicines have reached a value of 100

million dollars a year. About 60% of this is crude herbs (to be manufactured into

products outside India), about 30% is finished product shipped abroad for direct

sales to consumers, and the remaining I 0% is partially prepared products to be

finished in the foreign countries. (Dhannananda, 2004).

1.2 Regulatory control of herbal medicines world-wide

The World Health Organization (WHO) has reviewed the regulatory control of

hcrhal medicines in 50 countries and summarized the wide differences in the

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---;-----:----:--:---c----:-----~~~ approach of regulations between these countries. Herbal products are well

established as phytomedicines in some countries, whereas in others their

therapeutic claims are not allowed and regarded as foods.

In Australia, Complementary medicines, including herbal medicines are

regulated under therapeutics goods legislation 1989. For the purpose of labeling

requirements, herbs are included in the list of Australian approved names for

pharmaceutical substances, which is published by the Therapeutic Goods

Administration. Herbal medicines are regulated as drugs in Canada and must

conform to labelling and other requirements as set out in the Food and Drugs Act

and Regulations (Hoppe, 1981 ).

Herbal remedies used for minor self-limiting conditions are allocated a Drug

Identification Number based on pharmacological rationale. In order to facilitate

registration of products containing herbs, a series of Standardized Drug

Monographs has been prepared. The Drugs Directorate has provided guidelines

for the manufacture and sale of botanical products which fall into three

categories: food supplements (no DIN required, no therapeutic claims);

phytopharmaceuticals with full drug status (approved therapeutic indications,

approved dosage, efficacy supported by scientific evidence, DIN required);

traditional herbal medicines (self-medication only, efficacy supported from the

herbal literature, approved therapeutic indications and dosage)(Leung, 1980). In

November 2001, the Natural Health Products Directorate published a series of

further developments on the working draft of the proposed regulatory framework

for Natural Health Products (Mabey, 1988). Product licences, compliance with

good manufacturing practice (GMP), and a definition of natural products are

currently being subject to a review procedure.

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Tire m'jodty ofmOOidmll h~b• ~d thoiT prodoot• 're regol,.od lo lh::Jl foods or food additives, and most of the regulatory action has been concerned

with safety (Hoppe, 1981). In 1990, the Food and Drugs Administration (FDA)

reported on over-the-counter drugs, including herbs (Martindale, 1999). Some

herbs (e.g. cascara, senna) were pronounced safe and effective, but the majority

were not. Overall, 250 herbs, primarily based on their use as food additives, i.e.

flavours and fragrances, were designated GRAS (Generally Regarded As Safe)

status; however, this does not mean that such herbs (e.g. ginger, liquorice) are

FDA-approved for therapeutic purposes. In 1994, the Dietary Supplement Health

and Education Act (OSHEA) became law. In theory, this Act allows

publications, book chapters and scientific reports to support the sale of dietary

supplements (Martindale, 1999). Although medicinal claims cannot be made for

such products, labelling may describe effects on general well-being. OSHEA

and its regulations seek to define herbs in the context of the modem market

place, but they can only be regarded as an interim legislative step (Martindale,

1999).

In India, the first National Health Policy 1983, mentions that India's rich

tradition of health care should be included in national programmes. The National

health Policy states that TM experts and medicines should be used to stabilize

the population. The department of A YUSH, which started in 1995, regulates TM

programmes. There are separate directorates of Indian systems of medicine and

homeopathy in state governments. The government of India has an explicit and

separate policy for Indian (traditional) medicine since 2002.Traditional medicine

is also governed by the Drugs and Cosmetics Act, 1940. Licensing of

manufacturers and the provisions of the Drugs Act arc implemented by the state

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--------;::;:~~~~~ governments. GMPs are mandatory smce 2002, although not exactly as

recommended by WHO, but still they are based on them. Central and state

governments are impressing upon manufacturing units to comply with GMP

norms and to ensure quality standards.

1.3 WHO regulation of herbals

1.3.1 The WHO traditional medicine programme (WHO, 1998)

The World Health Assembly (WHA) has adopted a number of resolutions

drawing attention to the fact that a large section of the population in many

developing countries still relies on traditional medicine, and that the work force

represented by traditional practitioners is a potentially important resource for

primary health care. In 1991, the Director-General of WHO, in a report to the

forty-fourth WHA, emphasized the great importance of medicinal plants to the

health of individuals and communities. Earlier, in 1978, the thirty-first WHA had

adopted a resolution (WHA31.33) that called on the Director-General to compile

and periodically update a therapeutic classification of medicinal plants, related to

the therapeutic classification of all drugs; subsequently, resolution of

WHA40.33, adopted in 1987, urged member states to ensure quality control of

drugs derived from traditional plant remedies by using modem techniques and

applying suitable standards and good manufacturing practices; and resolution

WHA42.43, of I 989, urged member states to introduce measures for the

regulation and control of medicinal plant products and for the establishment and

maintenance of suitable standards. Based on the relevant WHA resolutions, the

major objectives of the traditional medicine programme arc to facilitate the

integration of traditional medicine into national health care systems; to promote

the rational usc of traditional medicine through the development of technical

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-g-u.,-id;-e-;cli:-n_e_s-an-d-;-i:-n-:-te-m-at:-;-io_n_a-;l-s-ta-n"""'d:-a-rd:-s--:-in-t7h-e--::fi:-e-:-ld-:----o-f::-:-h-e-=rb-a7l-m-e"""""dioi"< J acupuncture; and to act as a clearing house for the dissemination of information

on various forms of traditional medicine.

In resolution WHA42.43 (1989), the health assembly urged member states to

make a comprehensive evaluation of their traditional systems of medicine; to

make a systematic inventory and assessment (preclinical and clinical) of the

medicinal plants used; to introduce measures for the regulation and control of

medicinal plant products and for the establishment and maintenance of suitable

standards; and to identify those medicinal plants, or remedies derived from them,

which have a satisfactory efficacy/side-effect ratio and which should be included

in national formularies or pharmacopoeias.

1.3.2 Guidelines for the assessment of herbal medicines (WHO guidelines, 1991)

A WHO consultation in Munich, Germany, June 1991 drafted guidelines for the

assessment of herbal medicines (WHO/TRM/91.4), which were adopted for

general use by the 61h ICDRA in Ottawa, October 1991.

a. These guidelines contain basic criteria for the assessment of quality, safety,

and efficacy and important requirements for labeling and the package insert

for consumers' information.

b. The requirements for pharmaceutical assessment cover issues such as

identification, galenical forms, analysis and stability.

c. Safety assessment should at least cover the documented expenence of

safety and toxicological studies, where indicated.

d. The assessment of efficacy and intended use includes evaluation of

traditional usc through appraisal of the literature and evidence to support

the indication claims.

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----~~ 1.3.3 Guidelines for formulation of national policy on herbal medicines

(WHO guidelines, 1994)

The majority of the world population seeks treatment with traditional medical

practices, especially herbal medicine. The guideline was proposed to formulate

national policies on traditional medicines and to encourage co-operation between

member states. The aim of such national policies would be to develop regulatory

and legal reforms to ensure good practice, and to extend primary health care

coverage, while ensuring the authenticity, safety and efficacy of these medicines.

Main objectives include the recognition of traditional medicine as an integral

part of national health care systems, co-operation between modem and

traditional medicine, promotion of the rational use of products, introduction of

quality assurance systems, guarantee of regular supplies and to promote the

research and development of regulatory measures. It has been recommended to

countries that a National Expert Committee should be established, which would

be the appropriate authority to identify the steps and plans needed to formulate

national policy in this area and then to develop, direct and monitor the various

phases of its implementation.

1.3.4 Guidelines for the assessment of herbal medicines (WHO guidelines, 1996)

These guidelines for the assessment of herbal medicines are intended to facilitate

the work of regulatory authorities, scientific bodies and industries in the

development, assessment and registration of such products. The assessment

should reflect the scientific knowledge gathered in that field.

These guidelines defines the criteria for assessment of quality involving

pharmaceutical assessment of crude plant material, plant preparations and

finished products particularly using chromatographic fingerprints if no other

----~-------- --~~-----------~------------- ---

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~~------::----::----,-----,--:--~~ satisfactory criteria available; assessment of safety involving toxicological

studies with proper documentation and cited literature; assessment of efficacy

including the pharmacological and clinical effects of the active ingredients and

evidences to support the indications and Intended use of the guideline also

defines the information, which should be given to the consumers for use of the

preparation.

1.3.5 Guidelines for methodologies on research and evaluation of TM (WHO

guidelines, 2000)

The methodologies for research and evaluation of traditional medicine should be

based on the following basic principles. On the one hand, the methodologies

should guarantee the safety and efficacy of herbal medicines and traditional

procedure-based therapies. On the other hand, however, they should not become

obstacles to the application and development of traditional medicine.

The specific objectives of the guidelines are to. harmonize the use of certain

accepted and important terms in traditional medicine; summarize key issues for

developing methodologies for research and evaluation of traditional medicine; to

. improve the quality and value of research in traditional medicine and provide

appropriate evaluation methods to facilitate the development of regulation and

registration in traditional medicine.

1.3.6 WHO guidelines on GACP for medicinal plants (WHO guidelines, 2000)

The guidelines provide a detailed description of the techniques and measures

required for the appropriate cultivation and collection of medicinal plants and for

the recording and documentation of necessary data and information during their

processing. Good agricultural and collection practices for medicinal plants is the

first step in quality assurance, on which the safety and efficacy of herbal

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d. . I d d. ld d . I . ~I . ~ me Icma pro ucts uect y epen upon and will a so play an Important ro e m

the protection of natural resources of medicinal plants for sustainable use.

1.3.7 The WHO traditional medicine strategy (WHO, 2002-2005)

The WHO recognizes that the traditional use of herbal medicines refers to the

long historical use of these medicines and that they may be accepted by national

authorities. As a result of this view, the WHO traditional medicines strategy

2002-2005 was developed to review a framework for action for WHO and its

partners aimed at enabling traditional medicine/ complementary alternative

medicine (TM/CAM) to play a far greater role in reducing excess mortality and

morbidity especially among impoverished populations, which is one of WHO's

strategic directions for 2002-2005. Specific objectives in TM/CAM for 2002-

2005 are accordingly to support countries to integrate TM/CAM with national

health care systems, as appropriate by developing and implementing national

TM/CAM policies and programmes; to promote the safety, efficacy and quality

of TM/CAM by expanding the knowledgebase on TM/CAM, and by providing

guidance on regulatory and quality assurance standards; to increase the

availability and affordability of TM/CAM, as appropriate, with an emphasis on

access for poor populations and to promote therapeutically sound use of

appropriate TM/CAM by providers and consumers.

1.4 Ayurveda

Ayurveda is the science of health and healing practiced by ancient Aryans,

which is based on Atharvaveda, one of the oldest scriptures of Hindus, about

3000 years old. Ayurveda believes that various materials of vegetable, animal,

and mineral origin have some medicinal value. The medicinal properties of these

materials arc time tested and have been used for centuries in ayurvedic

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~~--~~ medicines to cure illness and/or help maintain health. Ayurvedic medicaments

are made from herbs or mixtures of herbs, either alone or in combination with

minerals, metals and other ingredients of animal origin. The metals, animals and

minerals are purified by individual processes before being used for medicinal

purposes. Impurified materials are not allowed to be used as medicine. The

object of ayurveda is to counteract the imbalance of three essential elements

vata, pitta and kapha (air, bile and phlegm respectively) which constitute the

tridosh from which the body originates. It is tridosha, which regularizes the

normal working of human body (Ayurveda Sar Sangrah, 2000). Ayurvedic

medicine is defined in Drugs and Cosmetic Act 1940, includes all medicines

intended for internal or external use, or in the diagnosis, treatment, mitigation or

prevention of disease or disorders in human beings or animals and manufactured

exclusively in accordance with the formula described in the authoritative books

of ayurvedic system of medicine specified in the first schedule of the act. In

ayurveda, the entire universe is composed of five basic elements called

panchmahabhuta and includes space (akasha), air (vayu), energy (teja), liquid

(jal) and solid (prithvi). The variable combinations of these principles make

tridosha in human body. These combinations are vata, pitta and kapha. They

have been interpreted in the following way.

Vata is composed of space and air (akasha and vayu). It is concerned with the

physical and mental activities which are activating or dynamic in nature. Vata

represents nervous system or nadichakra as per Ayurvedic terminology.

Pitta is composed of liquid and energy indicates biochemical/energy system. It

dominates nutritional and thermo genetic activities and also includes hormonal,

digestive and metabolic system.

J ·a!i.lation nf Prrparariotr rroccn and l"im:rrrrintim: nf wm(• A.lllnwlic r nrmulmiom

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----~~ Kaplla is composed of solid and liquid indicates various tissues and organs. It is

concerned with intergration of structural elements of the body.

Ayurvedic therapeutics is based on five pharmacological principles (panchsheel)

of durg. These principles are rasa, gun, virya, vipaka and prabhava.

1.4.1 Ayurvedic formulations (The Ayurvedic Formulary of india, 2000, 2003, Ayurveda

Sar Sangrah, 2000)

Ayurvedic formulations are those, which are prepared in accordance with the

formulae and procedures described in above authoritative texts. Different

solvents (menstrum) used in preparations of ayurvedic formulations are water,

oils, milk, ghee, cow's urine, etc. The use of sweetening agents, binding agents,

colourants, flavouring principles and other adjuvants are also very common in

ayurvedic preparations. With an objective of obtaining maximum therapeutic

benefit and making the formulation palatable, different pharmaceutical process

are prescribed in ayurveda. The important once are as follows:

Arka: It is the liquid preparation obtained by distillation of crude drugs in water

e.g.: Ajamodarka, Jatamamsyarka etc.

Churna: Fine powder of drug/drugs is known as chuma e.g.: Triphala, Trikatu,

Bhaskar Iavan chuma etc.

Dravaka: The liquid preparations obtained from lavanas or ksharas are known

as Dravakas. These are prepared by distillation process with or without addition

of any fluidse.g.: Sankha dravaka etc.

Ksharas: Alkaline (basic) substances obtained from the ash of drugs are known

as ksharas e.g.: Apamarga ksara, Kalyana ksara etc.

Lcpa: The preparation in the form of paste meant for cxtemal application on the

body is known as !cpa e.g.: Dasanga !cpa, Pathyadi !cpa etc.

-~~~-~-----~-------------------

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Vatika and Gutika: MOOk=ffiO ;, the form of illblet. of pill• = Jam~ li vatika or gutika e.g.: Bhuvneshvara vati, Drakshadi gutika, Prabhakara vati etc.

Netrabindu and Anjana : Netrabindu is processed by dissolving the specified

crude drugs in water or kasaya or honey and used as eye drops. Anjanas are very

fine powders of medicaments to be applied with netrasalaka e.g.:

Nayanarnrtanjana, Nagarjunanjana etc.

Sattva: Water extractable solid substance obtained from a drug is known as

sattva e.g.: Guduchi sattva etc.

Pisti: Pisties are obtained by triturating the drug with the specified liquids and

exposing the same to sun or moonlight e.g.: Pravala pisti, Mukta pisti etc.

Ghrita (Senehkalpa): Ghritas are the preparations in which ghee is boiled with

the prescribed quantity of the decoction (kasaya) and fine paste (kalka) of the

drug as specified in the formula e.g.: Amrta ghrta, Eladi ghrta, Tiktaka ghrta etc.

Taila: Tailas are preparation in which taila is boiled with prescribed decoction

according to the formula e.g.: Narayana taila, Triphaladi tailam etc.

Bhasma: The powdered form of the substance obtained by calcinations of

metals, minerals of animals products by a special process in closed crucibles or

in pits covered with cow dung cakes (puta), are known as Bhasma e.g.: Louha

bhasma, Vajra bhasma, Tarnra bhasm, Pravala bhasma etc.

Rasa-Yoga: The medicinal preparations containing mineral drugs as their main

ingredients in the form of powder or pills are known as Rasa-yoga e.g.:

Amlapittantaka rasa, Karpura rasa, Gangasundra rasa,

Fanta: These are prepared by pouring hot water over the dry crude drugs e.g.:

Kutja fanta.

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----~~ Nikadha: The residue or marc obtained in Kadha or Kashaya is treated with

boiling water and strained. The filtrate thus obtained is known as Nikadha.

Ghana-saar : The decoction obtained in the preparation of kadha or nikadha is

further evaporated to solid mass is known as Ghana-saar.

Kalka: When fresh or dry crude-drug is powdered and pounded with water hot

or cold to give a very fine paste then it is termed Kalka. e.g.: Apamarga kalka

Swarasa: When the Kalka is pressed in between the palms of the hand or is

expressed through the layers of cloth to produce a thick juice then it is called as

swaras. (Swa-rasa).

Asava and Arishta: Asava and Aristas are the medicinal preparations processed

by soaking the drug in the powdered forms or in the form of their decoction

(kasaya), in a solution of sugar of jaggery (Gur), for a specified period of time.

During soaking, it undergoes fermentation generating alcohol and in process

facilitating extraction of active constituents contained in the drugs Alcohol so

generated also serves as a preservative in the product e.g.: Lohasav, Kumariasav,

Dasmularistha, Draksharistha etc.

Nassyas: These are the nasal preparations and may be very fine powders,

aqueous or oily preparations.

Avaleha or leba and paka :Avaleha or Leba is a semisolid preparation of drugs

prepared by addition of sugar, jaggery (gur) or sugar candy and boiled with

prescribed drug-juice or decoction e.g.: Vasavaleha etc.

1.5 Standardization of Ayurvcdic formulation (WHO, 1996; 1998)

Standardization is the process of agreeing on technical standards. A standard is a

document that establishes uniform engineering or technical specifications,

criteria. methods. processes. or practices. The standardization of ayurvedic

r "a/idation (If Preparation Prt•CCH and f"ingaprintinr: of'""'~. tyun·(·dic l"'nmrulation~

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~---:---------:--~~~ preparations have many characteristic features and needs careful

considerations.In ayurvedic formulation, standardization refers to providing

processed plant material that meets a specified concentration of a specific marker

constituent.

Quality of raw materials is essential for the manufacture of standard drug. Raw

materials purchased from the market vary in quality almost from batch to batch.

Different manufacturers use different methods for their formulations. Thus, the

preparation will also vary being dependent on the nature of the method used.

Further from the academic standpoint, the knowledge of contents, constituents of

drugs and their uses always enrich the ayurveda materia medica and pharmacy.

Therefore it is necessary to give priority to the work of standardization of

ayurvedic research. Standardization of ayurvedic formulation is rendered

difficult because of specific methods are not available for identification of raw

materials, there are no standard uses prescribed in literature for finished products

and vaidyas in different states of the country had been selecting raw materials

and preparing drugs according to their own established methods.

The important stages of standardization of ayurvedic formulations are

I. Standardization of raw materials.

2. Standardization of preparation process

3. Standardization of the finished product

1.5.1 Quality control of traditional/ herbal formulations

The quality of phannaceuticals has been a concern of the WHO since its

inception. Quality Control is a term that refers to processes involved in

maintaining the quality of validity of a manufactured product. The safety and

efficacy of herbal medicines is closely correlated with the quality of the source

I ·alidatimt (If frrrllfatiml ProcC\\ and /lngcrprimim: of ~omc. tyun t•dic Fonrwlation~

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~~=-----:-:------~~~ materials used in their production. The quality of source materials is, in its tum,

determined by intrinsic factors (genetic) and extrinsic factors (environmental

conditions, cultivation and harvesting, field collection and post

harvest/collection transport and storage). Therefore, it is very difficult to perform

quality controls on the raw materials of herbal Drugs (Cardellina, 2002).

Therefore, production of quality traditional medicines has become a challenge to

regulatory authorities, scientific organizations and manufacturers.

1.5.1.1 Quality control methods

Methods currently utilized in evaluating herbs, their extracts and formulations

include organoleptic, microscopic, physical, chemical, chromatographic,

fingerprinting, microbiological, and biological methods.

1.5.1.1.1 Macroscopic analysis (WHO, 1998a)

Medicinal plant materials are categorized according to sensory, macroscopic and

microscopic characteristics. It is the first step towards establishing the identity

and degree of purity of such materials and should be carried out before any

further tests are undertaken. Visual inspection provides the simplest and quickest

means by which the identity, purity and possibly quality can be established.

Macroscopic identity of medicinal plant materials is based on shape, size, colour,

surface characteristics, texture, fracture characteristics and appearance of the cut

surface. However, since these characteristics are genuine material, it is often

necessary to substantiate the findings by microscopy and or physicochemical

analysis.

1.5.1. 1.2 1\licroscopic analysis (WHO, 1998a)

Microscopic evaluation allows more detailed examination of a drug and it can be

used to identify organized drugs by their histological characters. It also covers

f 'a/idmion flj Prcraration Prncc\fi and llnr:crpn'ntin.r: nfwml' Arun,•dic /'omwlatiom

18

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""'' of'""'""''"" by """"""" of oh=k& "'"' to •=II qow<i <i~ "' drug• I j in powdered form or to histological sections of the drugs. The characteristics of

cell wall, cell contents, trichomes, fibres, vessels, measurement of sizes of

tissues etc can be studied in details.

1.5.1.1.~.1 Quantitative microscopic analysis (The Pharmacopoeia of India, 1966)

In addition to the simple measurement of the sizes of tissues, cells and cell

contents, it is possible to estimate the % of foreign organic matter in powdered

drugs by a lycopodium spore method. Other microscopic determinations, which

may be useful in certain cases, include vein-islet number, vein termination

number, palisade ratios, stomatal numbers and stomatal indices.

1.5.1.1.3 Physicochemical analysis (The Pharmacopoeia oflndia, 1966)

Physical standards should be determined for herbal drugs. These are rarely

constant for crude drugs but may help in evaluation specially with reference to

moisture content, specific gravity, density, viscosity, solubility in different

solvents, ash value, extractive value, volatile oil content, foreign matter, acid

value, Iodine value, saponification value, unsaponificable matter, acetyl value,

ester value, crude fibre, swelling index, melting point, refractive index, foaming

index etc.

1.5.1.1.4 Chemical methods

Quality of drugs should meet the standards related to safety, potency and

efficacy. The aim of quality control is to evaluate whether the sample of drug

complies with the appropriate specifications. These tests involve chemical

analysis of active constituents and may vary from single entity or combination of

several potent drugs in formulation, finished product etc. These methods are

designed to set the permissive limits of tolerance for the product when it reaches

··alidation fJj rrrraration FrocrH and flngrrJ'dlltim: nfwme. h un·l'dic Tomwlatiom

19

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~~~~~~ the patient. Official Pharmacopoeial standards are designed considering all

known methods of manufacture, safeguard against the varying standards of

purity, impurity patterns, stability etc.

1.5.1.1.4.1 Volumetric methods

In volumetric methods, assay is based on the measurement of volume of solution

of known strength that is required to react completely with the substance to be

analyzed. On the basis of this measurement of volume, quantity of pure

component is estimated in this method. This method is classified in to different

types depending upon the type of reactions involved in the reaction (Mendham et

al., 2002).

1.5.1.1.4.1.1 Neutralization titration

Neutralization or aqueous acid-base titrations are usually reactions in which H+

in solution is titrated by OH- and this can be applied regardless of whether strong

acids, strong bases, weak acids, weak bases, weak acid salts, weak base salts in

the titration reaction (Mendham et al., 2002).

1.5.1.1.4.1.2 Non aqueous titration

This technique has now occupied a prominent place in analysis of drugs and

chemicals. The non aqueous titration has replaced the aqueous titration for many

substances which are either too weekly acidic or too weekly basic. It is based

upon the Bronsted-Lowrey and Lewis theory of acid and base and the nature and

influence of leveling effect of non-aqueous solvents on substances (Mendham et

al., 2002).

1.5.1.1.4.1.3 Precipitation titration

Precipitation is the combination of two ionic species to form a very insoluble

product. Precipitation of this product forces the reaction to completion, although

J ·alidation nf Preparation ProccH an,f rim:crl'n.nting nf wmc Ayunwlic T nrmu/atinm

20

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~-~---~~ such reactions are not uncommon. Their use in titrations is limited because not

all precipitation reaction meets the specifications. For titrimetric analysis the

precipitate must be practically insoluble, the precipitation reaction should be

rapid and quantitative, the results should not be hampered by adsorption effects

and it must be position to detect the equivalent point during titration (Beckett

and Stenlake, 2000).

1.5.1.1.4.1.4 Oxidation reduction titration

These reactions are the chemical processes in which a change in valency of

reacting elements or ions takes place. The reaction is thus a process involving

the transfer of electrons from one element or ion to another resulting in the

change of valency of reacting atoms or ions. An oxidation-reduction indicator

marks the sudden change in oxidation potential in the neighborhood of the

equivalence point in an oxidation-reduction titration. The ideal redox indicator

will be one with an oxidation potential intermediate. A sharp, readily detectable

colour change occurred at the end point (Jerry, 1992).

1.5.1.1.4.1.5 Complexometric titration Complexometric titrations are those

reactions in which simple metal ions are transformed into complex ion by

addition of the reagent which is known as ligand. The complex formed is stable

and water-soluble. Disodium ethylenediamine tetra-acetic acid (EDTA) is very

versatile complexing agent (Beckett and Stenlake, 2000).

1.5.1.1.4.2 Gravimetric methods

Gravimetric analysis or quantitative analysis by weight is the process of isolating

and weighing the compound of knovm composition i.e. purest form. The

separation of compound is affected by number of ways like precipitation,

volatilization electro analytical etc. In these first two methods are important in

21

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~~:--:---:--:--------:------:---~~ practice. Although gravimetric analysis is time-consuming, the constituent may

be examined for the presence of impurities and correction (Agarwal and

Agarwal, 1979).

1.5.1.1.4.3 Spectroscopic analysis

Spectroscopic analysis is concerned with the capacity of certain molecules to

absorb electromagnetic radiation at specific wavelength. This absorption or

emission is associated with changes in the energy states of the interacting

chemical species, and since each species has characteristic energy states,

spectroscopy can be used to identify the interacting species. Technique most

commonly employed in analytical field includes ultra violet, visible infrared and

atomic absorption spectrophotometry. The radiations utilized in spectroscopic

analysis can be roughly divided according to wavelength into the ultraviolet

(185-380 nrn), the visible (380-780 nm), the near infra red (780-3000 nm), and

the infra red regions (3-40 Jlm) (Willard eta!. 1986).

1.5.1.1.4.3.1 Colorimetric analysis

Colorimetry is concerned about the measurement of light in the visible region.

The basic principle of colorimetric analysis is the measurement of color of a

solution and its correlation to the determination of its concentration. The

measurement is made by comparing or reading the colour produced for the

substance with the same colour produced by a known amount of standard

material under well-defined conditions (Willard et a!. 1986).

1.5.1.1.4.3.2 Fluorescence analysis

When certain molecules are exposed to the electromagnetic radiation they

exhibit fluorescence. Fluorescence is a process of re-emission of radiant energy

absorbed in the form of visible light. In this process the light emitted is always of

l"a!id.Jtion (If Prrpararion Proccn and rinr:crpn·minr: of 5nmt. fyunw/ic r flrnmlatiom

22

INTRODU

high" ~wiffigili Jhm <hru •hrorl>ol. lo fl=•=re, 'b"'""'" md =i~ioo I j of light takes place in very short time (10-12 to 10-9 seconds). Ifthere is delay in

the emission of light then the phenomenon is called as phosphorescence. Both

these processes of re-quantitative determination of fluorescence are the basis of

fluorimetric analysis (Braun, 1987 and Willard et al. 1986).

For the quantitative studies their should be a definite relationship (linear)

between the concentration of species and the fluorescent intensity, which is

emitted such a relationship exists for very dilute solutions and fluorescent

intensity.

1.5.1.1.4.3.3 Infrared spectroscopy

The infrared absorption spectroscopy is concerned about the absorption of

infrared radiation by a molecule and exhibits characteristics absorption spectra.

Infrared spectrophotometer is a very important tool used in qualitative

identification and quantitative estimation of many drugs and chemicals. The

instrument is practically useful in pharmaceutical industry in identification of

drugs and detection of impurities (Bhal and Bhal 2001, Willard et al, 1986).1n

the infra red spectrum the fingerprint region (7-11 flm) is utilized to authenticate

and recognize many functional groups. British Pharmacopoeia involves

comparison of IR spectra of phytochemicals with European Pharmacopoeia

Chemical Reference Substances (EPCRS) as a test of identity (Evans 1996).

1.5.1.1.4.3.4 Nuclear magnetic resonance spectroscopy

NMR is designed for structural determination of organic compounds of natural

or synthetic origin. Use of H1 NMR spectroscopy and C13 NMR spectroscopy

have been described for assay of compounds described by TLC and HPLC and

confim1ation of certain groups and radicals position. It is concerned with the

J "a/idatimr (Jf Prt'raration rr(J('('H and llnr:crprilllin_r: (}I \(}nr(' A) un·cdic l"nmwlatiom

:23

INTRODU

~~ -:-ab;:-s:--o-rp---;:ti_o_n_o-;;f-c_e_rt-:-a-=-in_e_n_e_r_g_y--;-b-y-sp--:i-nn-I:-. n_g_n_u_c::-Ie-=i---=-in_a_m_a_g_n_e""ti:-c----;:-field when

irradiated by certain energy radiation perpendicular to it. Thus it permits

identification of atomic configurations in a molecule. Absorption occurs when

nuclei undergoes transitions from one alignment in the applied field to the

opposite one. The NMR spectroscopy enables us to record differences in

molecule and it also enables to know the positions of these nuclei within a

molecule. It enables to deduce different kinds of environment present in a

molecule and also about the atoms present in neighboring groups (Robert and

Francis, 2002, Willard et a!. 1986).

1.5.1.1.4.3.5 Mass spectroscopy

Like other spectroscopic techniques has been used as a routine method for

dealing with many analytical stable isotopes, examining ionization phenomena

studying free radicals etc. The mass spectrometer is an instrument in which the

substance in gaseous or vapour state is bombarded with a beam of electrons, to

form positively charged ions (captions) which are further sorted according to

their mass to charge ratio to record their masses and relative abundances. Both

positive and negative ions can be studied using mass spectrometer but usually

positive ions are analyzed since they are produced in large amounts as compared

to negative ions. The mass spectrum is a plot of relative abundance versus mass

to charge ratio. Each line upon the mass spectrum indicates the presence of

atoms or molecules of a particular mass (Willard et a!. I 986, Mendham et a!.

2002).

1.5.1.1.4.4 Methods for development of chromatographic fingerprints

Chromatographic methods represent the most useful and powerful technique for

the separation of components of a complex mixture. Because of the rapidity and

24

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~~--,------------~~~ effectiveness, chromatography has been used in all the fields particularly in

chemistry, biology, medicine, dyes, forensic departments and clinical studies

with much advantage over other methods. A notable advantage of

chromatographic methods is that they are relatively 'gentle' methods and

decomposition of substances does not occur. One more advantage is that the

separations can be carried out in micro or semi micro scale i.e. a very small

quantity of mixture is required for analysis. The method used primarily for the

separation of the components of sample, in which components are distributed

between two phase, one of them is stationary while the other moves. The

stationary phase may be a solid or a liquid supported on a solid or a gel and may

be packed in a column spread as a layer or distributed as a film. The mobile

phase may be gaseous or liquid. Though fundamentally it is separation

technique, it provides identification of compounds and also the quantitative

estimation. It finds application in analysis of varied types gases and pollutants,

petroleum and petrochemicals, oils and fats, food and flavors, drugs and

vitamins, steroids and alkaloids, blood and serum, pesticides and fungicides,

radioactive isotopes and a number of miscellaneous purposes.

1.5.1.1.4.4.1 Column chromatography

The basic principle underlying in adsorption chromatography is adsorptions of

component at the solid liquid interface. For good separation, the components of

should have different degree of affinity for the solid support i.e. adsorbent and

the interaction between adsorbent and component should be reversible. The

component having strong adsorption for column material is held up while that

component having less affinity moves do\m the column at faster rate as the clute

passes through the column (Mendham ct a!., 2002).

··olidation of Preparation rroccH and fingcrprintin.r: of fi(lf71('. tyun·,·dic r omwlatiom

25

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----~~ 1.5.1.1.4.4.2 Partition column chromatography

In partition column chromatography the solid adsorbent is replaced by a packing

material comprising as a support material coated with a stationary phase. The

stationary phase should be immiscible or at the most sparingly miscible with

mobile phase. In this technique the solute gets distributed itself between the two

phases depending on it's partition coefficient (Sharma, 1992).

1.5.1.1.4.4.3 Thin layer chromatography

Thin layer chromatography (TLC) is a simple and rapid method carried out using

thin layer of adsorbents on plates. TLC not only combines the advantages of

paper and column chromatography but in certain aspects it is found to be

superior to either method. As various materials of different adsorptive power are

used in TLC, the separation may result due to adsorption or partition or by both

phenomenon depending upon the nature of adsorbents used on plates and solvent

system used for development (Mukharjee, 2002).

1.5.1.1.4.4.4 Paper chromatography

Paper chromatography is the technique in which the separation of an unknown

substance is mainly carried out by the flow of solvents on the specially designed

chromatographic paper. In this case the solvent goes up by capillary action and

the separation is effected by differential migration of the substance due to

difference in distribution coefficients. The theoretical principle involved m

separation by paper chromatography is largely by partition coefficient.

The results are represented by Rr value which represent the movement or

migration of solute relative to the solvent front. This is indicating position of

migrated spots on chromatogram. R indicates the fraction of the solute molecules

in a solvent at any specified time (Sham1a, 1992).

J ·alitfation of rrcrnration Prncnt and llm:crpn·nlin.f! ()/ \Onlt!. trun,·Jic rormulatiom

26

INTRODU

----~~ 1.5.1.1.4.4.5 Ion exchange chromatography

The ion exchangers behave as a porous network, which carry a surplus electric

charge is compensated by the ions of opposite charge. When the ionization takes

place they are exchanged with the ions, which migrate into the solution

(Mukharjee, 2002).

1.5.1.1.4.4.6 High performance liquid chromatography

The obvious way to increase the flow rate and get efficient separation is to force

the liquid by a positive displacement pump or by gas pressure. This versatility

can be achieved by making certain modifications in columns and by using

smaller diameter and smaller surface area of column particles and by using other

suitable packing structure. HPLC is a high resolution and high-speed liquid

chromatography. It has several times more resolving power than open column

liquid chromatography. Hence it is used for speedy resolution of complex

mixtures (Edwards and Cobb, 1966).

1.5.1.1.4.4. 7 Gas chromatography

Gas chromatography is a widely used technique for separation of gaseous and

volatile substances, which are difficult to separate and analyze. It is a rather

simple and inexpensive method, generally efficient in regard to separation. The

gas as a moving phase is passed through a column containing adsorbent or a

liquid adsorbent supported on an inert solid. Thus adsorption or partition is

possible. In gas solid adsorption chromatography (GSC) the components of the

mixture distribute themselves between the gas phase and the adsorbent and the

separation is due to the difTcrcnces in adsorptive behavior. While in gas liquid

chromatography (GLC). the components of mixture distribute themselves

between gas phase and the stationary liquid phase according to their partition

J ·alidatimr of Prrparation ProaH and Tln_r:rrprintin.:: of.\nmt•. trun-,·1/ic ronnulatiom

27

INTRODU

ffi . Th ld fi . I b . .d . ~h ~ coe tctents. e so 1 unctwns on y as a support for t e hqm statwnary p ase,

enabling it to present a large surface to the gas. The GLC has a much greater

application in the analysis of the organic compounds, which has a measurable

vapour pressure at the temperature employed (Ewing 1995, Braun 1987).

1.5.1.1.4.4.8 Hyphenation procedures In the past two decades, combining a

chromatographic separation system on-line with a spectroscopic detector in order

to obtain structural information on the analytes present in a sample, has become

the most important approach for the identification and/or confirmation of the

identity of target and unknown chemical compounds.

1.5.1.1.4.4.8.1 GC-MS and herbal medicines

Mass spectrometry is the most sensitive and selective method for molecular

analysis and can yield information on the molecular weight as well as the

structure of the molecule (Zhang, et al. 2003 ). Combining chromatography with

mass spectrometry provides the advantage of both; chromatography as a

separation method and mass spectrometry as an identification method. In mass

spectrometry, there is a range of methods to ionize compounds and then separate

the ions. Common methods of ionization used in conjunction with gas

chromatography are electron impact (El) and electron capture ionization (ECI).

With the GC-MS, people could produce not only a chromatographic fingerprint

of the essential oil of the herbal medicine but also the information related to its

most qualitative and relative quantitative composition (Li, 2003, Velasco, 2003).

1.5.1.1.4.4.8.2 HPLC-DAD, IIPLC-MS and others

HPLC-DAD has become a common technique in most analytical laboratories in

the world now. With the additional UV spectral information, the qualitative

analysis of complex samples in herbal medicines turns out to be much easier

l·a/illlltion of rrc-raration Proct'H and nn~crprintim: nf Wm(' A_llln-ctlic f(lrmu/atiom

28

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~---~~ than before, for instance checking peak purity and comparing with the available

standard spectrum of the known compound to the one in the investigated sample

(Zhang, et a!. 2003).

Moreover, combined HPLC-DAD-MS techniques take advantage of

chromatography as a separation method and both DAD and MS as an

identification method. DAD and MS can provide on-line UV and MS

information for each individual peak in a chromatogram. With the help of this

hyphenation, in most cases, one could identifY the chromatographic peaks

directly on-line by comparison with literature data or with standard compounds,

which made the LC-DAD-MS becomes a powerful approach for the rapid

identification of phytochemical constituents in botanical extracts, and it can be

used to avoid the time-consuming isolation of all compounds to be identified

(He, 2000).

1.5.1.1.4.4.8.3 Hyphenation of CE

The hyphenated CE instruments, such as CE-DAD, CE-MS and CE-NMR,

appeared in the past decades. The techniques have also quickly been used for the

analysis of the samples from herbal medicines (Strum, 1998, Hsieh, 1993). On-

line coupling of capillary electrophoresis to mass spectrometry and other

spectrometry allows both; the efficient separation of CE and the specific and

sensitive detection can be achieved. A "total analysis device" has been recently

demonstrated in the case of on-line HPLC-UV (DAD)-FT-IR-NMR-MS

analyses.

1.5.1.1.4.5 1\ficrobiological methods

Medicinal plant materials normally carry a great number of bacteria and moulds,

often originating in soil. While a large range of bacteria and fungi form the

J ·alidation fl[ Prrparation Prnccn and rim:crprinting of 'i(li,Jt! Ayun·cdic ronnulariom

29

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~~~~~~ naturally occurring microflora of herbs, aerobic spore-forming bacteria

frequently predominate. Current practices of harvesting, handling and production

may cause additional contamination and microbial growth. The determination of

Escherichia coli and moulds may indicate the quality of production and

harvesting practices. Therefore to ensure the safety and quality of the herbal

preparation, specific tests for microorganisms like Escherichia coli, Salmonella

spp., Pseudomonas aeruginosa, Pseudomonas aeruginosa are performed. In

addition the total viable aerobic count and test for aflatoxins are also performed

(WHO Guidelines, 2004).

1.5.1.1.4.6 Biological methods

Biological assays (bioassays) are prescribed where the potency of a drug or its

preparation carmot be adequately determined by chemical or physical mean but

where it may be possible to observe the biological effect of the drug on some

type of living matter. The principle of such assay is to compare dose of the

sample' being tested produces the same biological effect as a given quantity of a

standard preparation. It is also important that conditions, under which the sample

and the standard preparation are tested, are identical in all respects of time,

environmental factor and biological media used (Mukherjee, 1996).

1.5.1.1.4.7 Stability studies

Shelf life of any medicine can be defined as the time period or duration up to,

which it is expected to retain its active ingredients i.e. 90% of label claim when

stored at recommended condition. Every product has its definite shelf life which

depends on various factors such as the stability of its active ingredients,

manufacturing process, interaction between active and inactive ingredients,

dosage form container closure system, environmental conditions encountered,

J ·a!id.ttion fJ/ Prrrararion PrflCt'H and rirrgrrpn·nring fl/ wmc -i.lun·t•olic r onrmlatiom

30

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---,------:--:--:-:-----:-----~~~~ contamination with metal and microbes etc., which cause the

degradation/spoilage of the product by initiating/accelerating certain chemical

reactions such as oxidation/reductions help to stabilize the drug and for

prediction of shelf life and its optimum storage conditions (Padiyar et a!., 1998).

1.5.2 Monographic approaches on quality control and standardization

The worldwide spread in the use of herbal medicines has produced various

attempts to develop herbal monographs to define identity and quality criteria as

well as therapeutic information. The German Commission E has been publishing

therapeutic monographs since 1984. The ESCOP has been developing

therapeutic monographs since 1991 and the WHO has initiated a project to write

monographs on widely used herbs around the world. In the United Kingdom

herbal monographs have been published since 1983 (Herbal Monographs, 1997).

The word monograph means a learned treatise dealing with one subject, usually,

in the scientific community, a plant or animal genus or particular species. In the

botanical world there are essentially three kinds of monographs: standards,

therapeutic, and combined. A standards monograph, the type usually found in a

pharmacopoeia, deals with information to determine the proper identity of a

plant genus or genus and species. This includes the taxonomy and nomenclature

of the plant (according to the linnean system of botanical classification and

nomenclature, including all synonyms), botanical description of the whole plant

and plant parts, microscopic descriptions of cellular structures of various plant

parts, and various chemical assays to determine identity and purity. USP

standards monographs arc examples of this type (Herbal Monographs, 1997). A

therapeutic monograph usually provides a definition of the plant drug (i.e. plant

part. nomenclature, etc.), a listing of key chemical compounds, uses and

J 'alidation nf Preparation froccn and rim:crpdntim: njJfJmt· Aruncdic l"flrmulariom

31

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~~---:----:-:-:--------:---~~ indications, contraindications (i.e., what conditions may be present to preclude

use of the herb), side effects, dosage (including per unit and total daily intake),

form of administration (i.e., the dosage in tea, tablet or capsule, liquid extract,

etc.), duration of use, interactions with other medications. The Drug Information

Division ofUSP has also produced two in this area (Herbal Monographs, 1997).

Then, occasionally there are combined monographs, in which both the standard

information as well as therapeutics is included. The WHO monographs and the

AHP monographs are of this type (Herbal Monographs, 1997).

Internationally several Pharmacopoeias including Indian Pharmacopoeia, British

Pharmacopoeia, Pharmacopoeia of Republic of China, Japanese Pharmacopoeia,

United State Pharmacopoeia, European Pharmacopoeia and many more have

developed monographs and quality control tests for few of medicinal plants and

some products made out of these herbs. Looking at increasing interest in the

natural raw materials, the U.K. founded British Herbal Medical Association

(BHMA) in 1964 to advance the science and· practice of herbal medicines.

BHMA published I" British Herbal Pharmacopoeia (BHP) in 1971, then in 1983

and 1996, giving monographs and quality control tests for some of the common

herbs and herbal products. Japanese standards for herbal medicines first

published in 1993. In India the first Ayurvedic Pharmacopoeia Committee

(APC) founded in 1962 and the Ayurvedic Formulary of India, first published in

1978, without giving preliminary standards for single drugs and compound

formulations due to lack of sufficient scientific data. In 1989, the first Ayurvedic

Pharmacopoeia of India came into account provided Pharmacopoeial standards

of some single drugs of plant origin (Chaudhri, 1996). USP is currently

accumulating a database of botanicals that arc approved in other national

-- ----------- -----~-----------------------------, 0/idatimr of ruparation ProcrH and llnr:crprintin.r: fi[Wnl<' A.llln·ctlic rnnnulatiom

32

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~~~~~~ pharmacopeias to make this information available to the USP General

Committee of Revision or its advisory panels in the event that questions

regarding botanicals arise during the 1995-2000 revision cycle (Herbal

Monographs, 1997). USP has moved forward and produced monographs that

creates a USP standard for various popular herbs in US commerce six draft

standards monographs for discussion purposes in the Pharmacopoeial Forum, a

bimonthly publication ofUSP

The German Commission E: The most widely acknowledged system for the

development of therapeutic monographs is that of the German Commission E.

Commission E is a division of the German Federal Health Authority (equivalent

to our FDA). Since 1978 when it was first organized and until it was terminated

by an amendment to the Medicines Act in August 1994, the Commission E

review published 330 monographs covering 324 herbs used in German folk

medicine, approving over 200 herbs as safe and effective for over-the-counter

drug use. The 330 monographs cover more than 80 percent of the herbal

medicine products in the German market (Keller, 1996). Commission E

monographs are geared toward therapeutic information on each herb, giving the

taxonomic nomenclature, botanical and chemical constituents, pharmacological

properties, and pharmacokinetics and toxicology (in more recent monographs),

as well as clinical information, including approved uses, contraindications and

side effects, special cautions, pregnancy and lactation information, interactions

with other drugs, dosage, overdosage information, special warnings, and so on, if

these are applicable.

One of most interesting developments in the area of herbal monographs is

ESCOP, an affiliation of national associations of phytothcrapy in Western

l·ali.latiml nf ruraratiml PwccH ami rirrr:crprintinr: nf .\(1111~. h unwlic rormulatiom

33

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~~ ;:E:-u-ro_p_e_w--;h_o_c_o"""'al:-e-sc-e--;d:-:-in---:lc::9::;8:;;9-as-a-re-s-u""lt-of-;;-:t""h-e """"'E:::-u_r_o-pe-an-7h-arm-onization

process. ESCOP has produced a series of herbal monographs in order to respond

to the increasing integration of what was formerly called the European Economic

Community (now European Union, or EU). The need for harmonized drug

monographs for herbs was recognized so that there could be standards for

botanicals popularly used in medicine and pharmacy in Europe. Unlike a

Pharmacopoeial monograph which focuses on standards for identity and quality,

ESCOP monographs, like those of the German Commission E, deal with

therapeutic aspects of each phytomedicine. Thus, ESCOP monographs include

approved therapeutic uses and contain much of the format like recent

Commission E monographs, including recommended dosage, side effects and

contraindications (if any), and other specific instruction, to help guide health

professionals, industry, regulators, and patients (Blumenthal, 1991). ESCOP

monographs do not deal with quality issues; this is covered in other

Pharmacopoeial monographs. For example, the European Pharmacopoeia

includes 60 monographs on herbal drugs. An additional 45 draft monographs

have been published in the Pharmeuropa for comments.

WHO monographs: In 1994 WHO began the compilation of a list of herbs that

are widely used in primary health care in various countries around the world in

order to consider developing monographs on each botanical (Herbal

Monographs, 1997). This move is consistent with WHO's previous publication

of its "Guidelines for the Assessment of Herbal Medicines," calling for the

development of monographs to help define the quality of herbal medicines used

as either "traditional medicines" and/or as approved nonprescription (over-the-

counter) drugs. A list of 30 plant species is being considered for the second

------------------------------J "a!idarimr nf rrcraration Pr(JCC\\ an.lli"m:rrrrinrin.r: nf \nmt•. fyun·cdic rormu/atiom

34

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~~~,------------,-~~ volume (Herbal Monographs, 1997). 25 monographs encompassing 28 plant

species on WHO's "Widely Used Medicinal Plants" list are scheduled to be

published in book form as Volume I in 1997. The herbs represent widely used

herbs and medicinal plants from various geographical regions.

American Herbal Pharmacopoeia: In 1977 Prof. Farnsworth proposed to the

American Society of Pharmacognosy (ASP) that it develop a Botanical Codex, a

listing of monographs describing and offering analytical standards for the herbs

and medicinal plants that were becoming increasingly popular in folk medicine

and commerce in the U.S. The AHP monographs are combined monographs;

they include much of the standard definitional and analytical information found

in conventional Pharmacopoeial monographs on standards, plus therapeutic and

clinical information. However, AHP goes a step further with the inclusion of the

Chinese and Ayurvedic energetic information not found in standard Western

pharmacopeias and clinical literature on herbs (Herbal Monographs, 1997).

The availability of various types of herbal monographs will fill up the current

need for information on quality control and safe, responsible uses of herbal

products. As the industry continues to grow at record levels, the industry's use of

the information in herbal monographs will ultimately help health consumers by

making available herbal products of increased quality and reliability.

l·alhlation (If frrparation Procrn m~;J firrr:aprinting of wmt-• A.Jun·t•dic romrulatiom

35

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---------·-----------~~ 1.6 Review of literature

Till today, there is no significant work has been carried out, except a few reports

and some of them are summarized below.

S.N. Formulation · • Scientific contribution References

I. Chyavanprash Developed High-performance liquid Govindarajan chromatographic method for the quantification of et.al. phenolics viz. gallic acid , catechin ,syringic acid 2007 and rutin

2. Vanga bhasma Studied a systematic characterization of the drug Wadekar et. a/. samples after various calcination stages by XRD, 2006 SEM and EDS analysis

3. Karpura Determined content of Fe & N by volumetric Saleem et. a/. shilajit analysis while content of Mg, AI & Ca by 2006 bhasma inductively coupled plasma emission spectroscopy

4. Chyavanprash Determined bio-efficacy by various biological Kasar et.al. methods like antioxidant study, microbial 2006 contamination study and fmally stability study of formulation according to ICH guidelines

5. Kurnariasava Standardization and determination of percentage of Elamthuruthy Aloin by HPTLC method et.a/.

2005 6. Prabhakara Vat Developed HPLC method for quantification of Bagul et.al.

i Gallic acid in the formulation & compared with 2005 marketed.

7. Svaras Developed reliable and feasible HPLC method with Scartezzini Bhavana diode array detection for the determination of et.a/.

ascorbic acid in Emblica fruit 2005 8. Chyavanprash Developed spectrophometric estimation of tannins Jain et.al.

by folin-denis method using 2004 phosphotungustomolybdic acid for the formation of colored complex

9. Kapardika Characterization of raw material, intermediate Bhagwat et.al. bhasm obtained during the process and final product by 2004

using powder XRD and IR techniques 10. Chyavanprash Performed high performance thin layer Rastogi et.a/.

chromatographic methods for quantitative analyses 2004 of different amino acids viz. alanine, valine and lysine

11. Jasad bhasm Characterization of Zinc oxide in raw material, Bhagwat et.a/. intermediate obtained during the process and final 2003 product by using powder XRD

12. Chyavanprash Analyzed extractive values of three batches of Jain et.al. chyavanprash in different solvent by 2003 spectrophotometrically for spectrum, overlain spectrum and linearity studies

13. Navayasa Performed standardization for estimation of piperine Eapen et.al. Chuma and em be lin content by HPTLC and iron content was 2002

estimated by U.V. Spectroscopic method. 14. Shanha Determined physico-chemical characters of Calcium Lalla ct. a/.

Bhasma Carbonate and studied safety and efficacy of the 2002 formulations

l'alidation (If Prt'raration Prnccu anrl lln.r:crrrimin,r: nf \Om<!. t.1 un-cdic /Omrulathm\

36

INTRODU

~! N

1 s. Khutajgan vati Evaluated quantitatively and qualitatively by Bhavsar et. a/. employing TLC and titrimetric methods for detection 2001 of alkaloid in Kurchi bark and Ativish root in formulation

16. Triphala Evaluated extractive values, ash value, Lalla et. a/. chum a phytoconstituent and some physical constants and 2001

compared it with similar formulations

17. Swama- determined microelements and metal content in the Mitra et. a/. bhasma bhasma by Infrared spectroscopy and atomic 2001

absorption spectrometry 18. chum a Reported stability studies and physico-chemical Padiyar et. a/.

protocols of chuma as ]JOr ICH guidelines 2000 19. Trikatu chuma Determined percentage of piperine by UV Sheth et.al.

spectrophotometric method 2000 20. General Presented standardization, quality control and quality Agrawal

Ayurveda assurance of Ayurvedic formulations et.a/. 2000

21. Vajraka Taila Developed techniques for Ayurvedic preparation and Dwivedi standardization of their, pharmaceutical/ et. al. manufacturing technique, physico - chemical 1999 parameters individually applicable, self-life of the product

22. Angya Reported Ayurvedic properties of its individual Giri et.al. vardhani vati constituents, its medicinal properties, diseases for 1999

which used, doses and type of vehicle to be used for various cases

23. Ksirabala Analyzed oil for determination of its pharmacopoeial Alam et.a/., tail am parameters 1998

24. Civanar Prepared in laboratory for standardization and Saraswathy Vembuk Kulit chemically analysis for identity and purity. The et.a/. Tailam physico-chemical data, TLC and HPTLC finger 1998a

printing profile were evolved for fixing standards and also identifY the plant drugs used in the medicine

25. Shatshakar Developed identification parameters for some Singh et. a/. Chuma Ayurvedic drugs using IR spectrophotometry 1997 Dashmularista Abhayarista Shitoplas chum a

26. Parthadhyarist Developed standardization parameters of Arjuna Rao et.al. a bark (Terminalia arjuna),Mridvika fruits (Vitis 1997

vinifera) and Madhuka flowers (Madhuca indica 27. Sinduradya Analyzed samples of Sinduraya taila according to Pandey et.al.

tail a textural references and standardized for their physico 1997 chemical constituents

28. Nasya Identified and standardized the Ayurvedic Nair et.al. Snehapana formulations 1997 Abhayanga

29. Vachalasunadi Detection of Acarus callamus, Curcuma longa Thankamma Bilvapatra Allium sativum and Aegle marmelos in formulations et.al. tail a by TLC methods 1996

30. Tribhuvan- Developed TLC method for confirmation of the Agrawal akini presence of main ingradicnt Vatsanabha Aconitum ct. a/.

chasmanthum 1996

l'a/idation nf Prrr(1rarion Procrn and lln.ccrprinting of,nmt A.1 urwdic romrulatiom

37

INTRODU

----~~ 31. Pinda Taila Detected the presence of drugs present in pinda Tai!a Hepsibah

that was prepared using Rubia cordifolia, et.a/., Hemidesmus indicus and vateria indica and beeswas 1996 byTLC

32. Polyherbal Standardized some marketed Ayurvedic anti-diabetic Purohit et al., formulations formulations 1995

33. Vacadi taila Detected Acorus calamus in Ayurvedic preparations Thankamm byTLC et.a/.

1995 34. Chyavanprash Standarded popular Ayurvedic adaptogenic Mahrotra

preparation and developed ethnobotany of its et.al. 1995 ingradients

35. Manasamitra Developed protocols for the isolation and separation Bhutani et. a/. vataka of the bioactive rich fraction from individual plants 1994

showing antistress, immuno-modulatory and memory enhancing Ayurvedic drugs and their standardizations

36. Gorocanadi Developed macroscopic, microscopic evaluations, Raina, et. a/. Vati quantitative chemical testes and chemcial finger 1993

printing by using UV /TLCIHPTLC/ techniques and fluorescence studies

37. Dashmula Standardized some Dashmula containing Alam et.al. formulations viz. Dasmularistha, Dashmula kwath, 1993 Dashmula taila containing Urcaria lagapodioides, Solanum xanthocarpum, Tribulus terristeris, Gemlina asiatica, Aegle marmblos, Premna integrifolia etc. and analyzed for total alkaloidal content

38. Tirikatu Reported physico-chemical parameters ofTirikatu Saraswathy churanam churanam consisting of Zingiber officinale, Piper et.al.,

nigrum, Piper longum havebeen 1993a

39. Bhringaraja Standardized Bhringaraja taila containing Eclipta Tiwari et.al., tail a prostana, Rubia cardifolia, Santalum album, Sida 1993

cardifolia, Berberis aristata 40. A sa vas Analyzed specific gravity, alcohol content, pH value, Dutta, et.al.

Aristas total solid content and sugar content in Asavas and 1992 Ghrita Aristas and R.I., Acid value, Iodine value in Ghrita Taila and Taila

41. Regulation of Discussed standardization Problems, realities and Katiyar et.al., Avurveda rationale of A vurvedic drugs: 1992

42. Bhasama, Developed quality control parameters of indigenous Tiwari et. a/. As a vas, pharmaceutical products and technique of 1992 Aristas, preparation ofbhasama, asavas, aristas and avaleha Avaleha has been demonstrated.

43. Shereeshadi Determined HPLC and RP-HPLC methods of Sharma et. a/. kashaya Vasicine in poly-herbal formulations 1992 Yastvadivati

44. Triphaladi Developed physico-chemical data of traditional drug Siddiqui Kvatham conatining harda (Terminalia chebula), balhera et.a/.,

(Tcrminalia belerica and amla (Emblica omcinalis) 1991 45. Swaskuthara Developed quantitative and qualitative characters of Shastry et.al.,

rasa Rasa along with the general parameters like L.O.D. 1991 Ash value, water and alcohol soluble extractive etc.

46. Ksara sutra Standardized Ayuncdic formulations used for the D" ivcdi treatment of fistula ct. a/ ..

1991

-. ·- -- .. ---------------------------- -----------· J 'a/idatinn fJ/ rrrraration PrfJCt"H and f7rtr;cf'{'rintin.r: of Hlfrl~ fyun·cdic romru/atiom

INTRODU

----~~ 47. Dasmula taila Identified and developed by TLC & PC of Dasmula Saxena et. a. I

taila by using suitable slvents and spray reagents 1990 48. Dasamulam Developed methods for standardization of Somanathan

kasayam Dasamulam kasayam for the comparison of market et.al. samples with laboratory sample 1989

49. Panchsakar Identified and standardized Panchsakar chuma Patel et.al. chuma 1986

50. Drakashrista Developed methods for standardization of Joshi et.al. Drakashrista 1986

51. Drakshadi Studied standardization ofDrakshadi Vati composed Alam et.al. Vati of Vitis vinifera (Draksha), Terminalia chebula 1985

(Pthya), and Sita (Sugar candy) 52. Dhanvantra Developed methods for standardization of Alam et.al.

Ghrta Dhanvantra Ghrta 1983 53. Chandraprabh Developed colorimetric method for standardization Alam et.al.,

a vati of Chandraprabha vati 1982 54. Chittiramoola Developed methods for standardization and also Anand el. a/.

thailarn determined physical and chemical characteristics of 1982 Chittiramoola thailam like refractive index, sp. gr., iodine value; acid value, saponification value and unsapouifiable matter

··a/idarion of Preparation rrr'CCH and fl~t.r:crrrintinr: nf Hlmt". fyun·cdic Tormulatiom

39

INTRODU

----~~ 1. 7 Research envisages and plan of work

In the last few decades there has been an exponential growth in the field of

herbal medicine. It is getting popularized in developing as well as in developed

countries owing to its natural origin and lesser side effect.Traditionalfherbal

medicines, which formed the basis of health care throughout the world since the

earliest days of mankind are still widely used, and have considerable importance

in international trade.

Ayurveda is an ancient and popular form of holistic medicine system originated

in India and now spreading worldwide. In Ancient time when the ayurvedic

medicines were developed, preparation, standardization and quality control of

ayurvedic formulation maintained by fully committed professionals. The

traditional ayurvedic text claims that ayurvedic formulation prepared according

to the method given in ayurvedic text will superior in quality, safety, and

efficacy. Process of preparation hold significant role in effectiveness and quality

of ayurvedic formulations. Process validation is "a documented program which

provides a high degree of assurance that a specific process will consistently

produce a product meeting its predetermined specification and quality

attributes". The quality of pharmaceuticals has been a concern of the World

Health Organization (WHO) since its inception. Many international authorities

and agencies including the European Agencies for the evaluation of medicinal

product and European Scientific Co-operation of phyto-medicine, U.S. Agencies

for Health Care Policy and Research, European Pharmacopoeia Commission,

Department of Indian System of Medicine and such have started creating a new

mechanism to induce a quality control and standardization of botanical and

ayurvcdic medicine. It is absolutely necessary to develop reliable analytical

J ·alidttlimr ''~ frcparatirm rr,.CC\t and ringcrpn·nring ll/ .~flm,; Ayun·cdic r ()nttulation~

40

INTRODU

---:;--;----;--~~~~~ methods in order to control the quality of herbal medicines. Fingerpnnt

technique was introduced and accepted by WHO as a powerful tool for

identification and quality evaluation of multi -component herbal/traditional

medicines. Ayurvedic formulations cover wide range of product from solid to

semisolid to liquid dosage forms. There fore the formulations were selected from

each dosage forms. The selected formulation for present research work has taken

by discursive attempts of studying various classical literatures. The identified

formulations are enlisted in the essential drug list declared by Central Council of

Ayurveda. The formulation was selected in the list are available in each

ayurvedic dispensaries from metro to micro interiors. The present work is an

approach to develop preparation process validation and fmgerprinting for some

formulations viz Arjunaristha (AJR), Vasavleha (VSL), Bhaskar Iavan chuma

(BLC), and Chyavanprash (CHY), which are official in Ayurvedic Formulary of

India.

l·a!idt1timr nf Prrraratirm Prnccu and 1/m:rrprintinr: ofwm<' Arun·<·dic rnrmu/atinm

41

INTRODU

----~~ 1.7.1 Candidate formulation

Arjunaristha

Arjunaristha is a well known ayurvedic formulation described in ayurvedic text

Charak-Samhita, Bhaisajyaratnawali and Ayurvedic formulary of India. It is a

potent ayurvedic medicine containing self-generated alcohol used as general

tonic for heart disease, lung diseases, loss of strength, immunomodulator and in

azoospermia. Aristhas are classical ayurvedic liquid dosage form made by

soaking the drugs (powder or decoction) in a solution of jaggery for a specified

period of time, which undergoes a fermentation process to generating alcohol,

thus facilitating the extraction of active principles contained in the drug(s). The

alcohol generated, also serves as a preservative.

Vasavleha

Vasavleha is a well known ayurvedic formulation prescribed in Ayurvedic

formulary of India and in classical ayurvedic texts Bhaisajyaratnawali, and

Charak Samhita. It is used for Asthma, cough, fever, angina pectoris and for

abdominal pain. Vasavleha falls under the category of Avaleha-paka group of

ayurvedic formulations. The main components are Piper longum and Adhatoda

zeylenica. Method of preparation for avaleha-paka group of ayurvedic

formulations is very tedious and plays a vital role in quality, and efficacy of any

formulations.

Bhaskar Iavan churna

The Bhaskar Iavan churna, a well known formulation described in Ayurvedic

formulary of India, traditionally used for digestive impairment, rheumatism,

dyspepsia, malabsorption syndrome, angina pectoris, disease of skin, splenic

disorder, oedema, asthma and constipation. Among all the ayurvedie medicine

J 'alit!ation nf Preparation rrnccu and llngc·rrrimim: nf \Onlt*. trunwlic rnrmulatiotl\

42

INTRODU

fO< tho ga<ro '""-""'' dl""'b'"" Bh""'M Ia- chama;, llia mo.t relabc&ol 11 by the ayurvedic practitioners of India. It falls under the category of Churna in

ayurvedic formulation.

Chyavanprash

Chyavanprash is a very commonly used ayurvedic medicine and health

supplement since centuries. It is reputed as antioxidant, nervine tonic, anxiolytic

having adaptogenic activity. In modern time it acts as immunomodulator,

memory enhancer, growth promoter in children's and digestive fire improver.

Administration of this r~uvenation therapy promotes intellect, memory, luster,

immunity to diseases, longevity, strength of sense organs, sexual excitement,

great stimulation of digestive and clarity of complexion. Chyavanprash falls

under the category of avaleha-paka group of ayurvedic formulations. Method of

preparation for avaleha-paka group of ayurvedic formulations is very tedious

and plays a vital role in quality, and efficacy of any formulations. The entire

Indian market for Chyavanprash stands about 50-60 tones, even though very few

quality control methods for its evaluation has been attempt. Till date no work

were carried out in the field of validation of preparation process for assuring the

quality and consistency with methods for fingerprinting of the most popular

ayurvedic formulation.

43

INTRODU

----~~ 1.7.2 Plan ofwork

The present study is an approach to develop, preparation process validation and

fingerprinting methods for some identified ayurvedic formulations that are

official in Ayurvedic Formulary of India. In order to achieve this objective

following studies have been undertaken:

>- Preparation of selected formulations and bifurcation of process m

different stages.

>- Validation of preparation process of different stage samples and finished

formulations.

>- Development of quality control parameter of formulation and their

validation.

>- Development of finger print method of formulation using sophisticated

instrument.

>- Evaluation of marketed formulation and comparison with the laboratorv

formulation.

>- Stability studies of laboratory formulation.

------------------------ ---·-----------' ·alidarimt fl[ Prrraratimt ProccH and riTt::crrrimin.~ of wmc Ayunwlic r flrmulatinn"i

44

INTRODU

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