CONTENTS

Serial Topic Page

no. no.

01. Acknowledgement 02

02. Abstract 03

03. SPAN Laboratories Pvt. Ltd. – A Company Insight 05

04. Products of SPAN Laboratories Pvt. Ltd. 08

05. Sales and Marketing Strategy adopted by SPAN Laboratories 13

Pvt. Ltd.

06. Ayurveda – An Overview 18

07. Scope of Ayurvedic Medicine 23

08. How the Ayurvedic Tradition became a system of Empirical 27

Medicine

09. Quality Control of Ayurvedic Drugs 30

10. Protocols for testing the quality of Oils, Paste, Tablets Powder 54

& Syrup

11. Results 88

12. Conclusion 94

13. Bibliography 99

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 ACKNOWLEDGEMENT
This project has been made possible with the help of a lot of people. My learning would be
incomplete if I don‘t acknowledge people who catalysed this process, Starting with the key
stone of the edifice Mr. Subhash Chandra (Managing Director) who obliged me by giving an
opportunity to undergo a short term summer training program under his esteemed guidance at
Span Laboratories Private Limited, Lucknow.

I would also like to thanks Mr. Prabhash Chandra (Production Manager) and Mr. Guddu
(Assistant) for their help during my training.

I would also like to extend my gratitude towards Mr. A. K. Saxena (Packaging Manager), Mr.
Swapnil Chandra (Marketing Head-OTC Division) and Mr. Saurabh (Assistant) for giving
their valuable time, guidance suggestions and critical comments which aided my learning
and work.
I would specifically like to thank all the employees of the Span Laboratories Private Limited
for the help and cooperation provided.

I also thank my friends Hemant, Sudhanshu and Ashutosh for their cooperation and support.
I would also like to thanks my parents and God without whose blessings it would not have
been so easy.

(Mudit Misra)

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 ABSTRACT
Medicinal plants constitute a source of raw materials for both traditional systems of medicine
(e.g. Ayurvedic, Unani, Homeopathy, and Siddha) and modern medicine. Nowadays, plant
materials are employed throughout the industrialized and developing world as home
remedies, over-the-counter drugs, and ingredients for the pharmaceutical industry. As such,
they represent a substantial proportion of the global drug market. Most rural populations,
especially in India, depend on medicinal herbs as their main source of primary health care.
Although most medicinal herbs are not, in their natural state, fit for administration,
preparations suitable for administration are made according to pharmacopeia directions. The
therapeutic potential of a ayurvedic drugs depends on its form: whether parts of a plant, or
simple extracts, or isolated active constituents.
Ayurvedic remedies consist of portions of plants or unpurified plant extracts containing
several constituents, which often work together synergistically. The ayurvedic drug
preparation in its entirety is regarded as the active substance and the constituents are either of
known therapeutic activity or are chemically defined substances or group of substances
generally accepted to contribute substantially to the therapeutic activity of the drug.
Phytochemical screening involves botanical identification, extraction with suitable solvents,
purification, and characterization of the active constituents of pharmaceutical importance.
Qualitative chemical examination employing different analytical techniques is conducted to
detect and isolate the active constituent(s). In general, all medicines, whether they are
synthetic or of plant origin, should fulfill the basic requirements of being efficacious and safe.
Ultimate proof of these can only be achieved by some form of quality testing. A defined and
constant composition of the drug is therefore one of the most important prerequisites for any
kind of clinical experiment.

Quality control for the efficacy and safety of ayurvedic products is essential. The quality
control of phytopharmaceuticals may be defined as the status of a drug, which is determined
either by identity, purity, content, and other chemical, physical or biological properties, or by
the manufacturing process. Compared with synthetic drugs, the criteria and the approach for
ayurvedic drugs are much more complex.

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Phytopharmaceuticals are always mixtures of many constituents and are therefore very
variable and difficult to characterize. The active principle(s) in phytopharmaceuticals are not
always known. The quality criteria for ayurvedic drugs are based on a clear scientific
definition of the raw material. Depending on the type of preparation, sensory properties,
physical constants, moisture, ash content, solvent residues, and adulterations have to be
checked to prove identity and purity. Microbiological contamination and foreign materials,
such as heavy metals, pesticide residues, aflatoxins, and radioactivity, also need to be tested
for. To prove the constant composition of ayurvedic preparations, appropriate analytical
methods have to be applied and different concepts have to be used in order to establish
relevant criteria for uniformity.

With many of these ayurvedic medicines we do not fully understand how they work. Nor do
we always know which component is pharmacologically active. Even though ayurvedic
remedies may be effective, do their benefits outweigh their risks? In some countries
ayurvedic remedies are sold as food supplements, thus evading safety regulations. Can
ayurvedic medicines save money? Not all plant-based medicines are cheap.

Even though global herbal resources have a great potential as natural drugs and are of great
commercial importance, they are very often procured and processed without any scientific
evaluation, and launched onto the market without any mandatory safety and toxicology
studies because there is no effective machinery to regulate manufacturing practices and
quality standards. Although some ayurvedic medicines are efficacious, there is
unquestionably a need for more reliable information, a demand that must be met adequately
by doctors, pharmacists, and other health care professionals.

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 SPAN LABORATORIES PRIVATE LIMITED – A Company Insight
Span Laboratories Private Limited is a GMP certified company. It is a leading name in the
field of pharmaceutical products. The company was established in 1996 at Gomti Nagar,
Lucknow. Thereafter, the company was incorporated on 6th April, 1996.

The company has been in the field of manufacturing and marketing of ayurvedic
pharmaceutical products since fourteen years. Span Laboratories carry a range of ayurvedic
products to suit every task great or small. Span Laboratories supply a range of liver
stimulants, products for digestive system, laxative drugs to suit every one. The company is
well equipped with large number of state-of-art machineries and a dynamic and dedicated
well trained workforce.

Span Laboratories is headed by Mr. Subhash Chandra, Mr. Prabhash Chandra and Mr. S.P.
Saxena as Directors. It is managed by a dedicated team of skilled professionals and highly
trained sales team.

The company is highly grateful to its team for its dedication and help. What we are is just
because of the support and dedication of our team. The Span Laboratories team consists of
internationally experienced sales executives, production managers and technology experts.
Our team works in co-ordination with each other, which are enthusiastic as well as
industrious in nature.\

Our Sales team is headed by Mr.Suhail Sahid, Mr.Syed Ifrahim and Mr.Jay Prakash
Saxena who have more than 15 years of experience in pharmaceutical industry. Each of them
possesses a specialized knowledge of industry. They carry out our global sales operations
rivigorously and with great spirit. The unique combination of our team add to the value of our
company and opens new perspectives for making sound decisions.

MISSION AND VISION

At Span Laboratories Private Limited our vision is to provide quality services to our
customers. Our goal is to achieve open rewarding workplace environment which enables our
colleagues to realize their full potential.

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Our Mission is to achieve excellence in all respect. We will act with honesty in dealing with
each other, our customers, our employees, our chemist and all other people who are in contact
with us.
Our vision revolves around quality production, teamwork, honesty, integrity and developing
environment in all aspect of our performances and business strategies.

PRINCIPLES AND CORE VALUES OF SPAN

 Ownership: Accept personal responsibility, and accountability to meet the business
needs.
 Passion for Winning: Be the leaders in area of responsibility, with a deep commitment
to deliver results.
 People Development: Our ethics are considered to be the most important asset. Value
addition is done through result driven training, and they encourage reward excellence.
 Consumer Focus: Give importance to customer needs and develop products to fulfil
them better.
 Team Work: Work together on the principle of mutual trust and transparency in a
boundary less organization. Team members are intellectually honest in advocating
proposals, including risks involved.
 Innovations: Continuous innovations in products and processes are the basis of our
success.
 Integrity: Achievement of business success with integrity is another important
principle. We believe in honesty with customers, business partners and also with each
other.

STRATEGIC INTENT
 Focus on growing core brands across categories, reaching out to new geographies,
within and outside India, and improve operational efficiencies by leveraging
technology.

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  Be the preferred company to meet the health and personal grooming needs of our
target consumers with safe, efficacious, natural solutions by synthesizing our deep
knowledge of ayurveda and herbs with modern science.
 Build a platform to make SPAN Labs. an indian as well as a global ayurvedic leader.
 Be a professionally managed employer of choice, attracting, developing and retaining
quality personnel
 Commitment towards environmental protection
 Provide superior returns, relative to our peer group, shareholders.

QUALITY POLICY
At SPAN, quality is a relentless commitment to continuous improvements in product, process
and systems to provides consistent quality products to meet our customer‘s requirement
worldwide. The management is fully committed to quality and ensures all resources to
accomplish this task.
Our Quality Objectives include:
 Focus on our customers and successfully meet their needs and requirements.
 Manufacture effective health care products at competitive prices and to improve the
quality of life of the common masses.
 Implement systems to ensure prevention of errors either than detection of errors.
 Ensure global competitiveness by striving to achieve current good manufacturer
practices (GMP).
 Ensure safety in all operations by working according to the system in all areas of
operations.
 Increase productivity and reduce wastage with in the organization.
 Provide appropriate training to the people to improve their skills and expertise, thus
strenghthening our commitment to the quality process.

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 PRODUCTS OF SPAN LABORATORIES
 DIGENORM SYRUP, CAPSULES & TABLETS

 EVACUA POWDER

8
 LIVGUARD SYRUP

 REGAIN SYRUP

9
 RHA OIL

 SENSATION CAPSULE

10
 SPANDRYL SYRUP

 SPANLIV DS SYRUP, TABLETS & CAPSULES

11
 UREE SYRUP

 U TONE SYRUP & CAPSULES

12
 SALES & MARKETING STRATEGY ADOPTED BY SPAN
LABORATORIES
In theory, the purpose of selling is to help a customer realize his or her goals in an economic
fashion. However, in reality this is not always the case. Customers can be influenced to
purchase a product or service that initially was not of interest to them. Some salespeople are
trained in the art of selling customers things they don't need.

Take for example the purchasing of a car: a consumer may have a set of cars in mind (called
an evoked set) that she feels match her needs, wants and budget. She may seek the advice of a
salesperson given that a salesperson can help her realize the right car given those criteria.

This can be a socially useful function; salespeople have specialized knowledge of products
that can help consumers make an informed decision. However, a salesperson may also talk a
consumer into purchasing a more expensive or perhaps larger car then she needs or can
afford. In this context, the salesperson may have usefully helped the customer re-evaluate her
needs, thereby establishing a new set of appropriate choices among which included the newer
or large car. This again would be a helpful and useful service provided by the salesperson.
However, it is sometimes the case that customers purchase a product or service that was not
initially intended and remains an inappropriate purchase after the fact. On the other hand, the
consumer in this scenario can be held partially responsible for the inappropriate purchase;
indeed, "A fool and his money are soon parted."

This dysfunctional behavior is encouraged by:

 Incentives from the manufactures of products or the companies of service providers to
salespeople to sell their products where other similar products offered by competitors
are offered.
 The incentive to sell a customer a product that is in need of being cleared out, despite
the fact that a customer may be better to wait for the new product.
 Incentives of salespeople to increase their total number of sales especially where
retailers keep track of sales or offer commission-based salaries.

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 DISTIRTUBTION CHANNEL OF SPAN LABORATORIES

Manufacturing Plant

Clearing and forwarding agent (different regions)

Stockist A Stockist B Stockist C

Retailers Retailers Retailers Retailers Retailers Retailers

C O N S U M E R S

The above diagram it shows channel of distribution of SPAN LABS Products, here first the
products are manufactured and from Manufacturing plants the packed goods are supplied to
Clearing And Forwarding Agents(C&FA) and from here the goods are then further supplied

14
to number of Stockiest or Distributors, from here goods reaches to large number of Retailers
and it is the duty of Stockiest to take orders from retailers and then supply the goods to them,
this work is generally done by stockiest salesman through ready stock or by taking orders
first and then placing the order. From here the goods finally reaches to Customers. Customer
purchases the product from retailers.

This was the basic Channel of Distribution used by SPAN LABS Products, now I will throw
light on each channel of distribution of SPAN LABS Products.

SUPPLY CHAIN MANAGEMENT:

Supply chain management starts before physical distribution: it involves procuring the right
inputs (raw materials, components and capital equipment), converting them into finished
products and dispatching them to the final destinations. The supply chain perspective can
help identify superior suppliers and distributors and help them improve productivity, which
ultimately brings down the company‘s costs.
A broader view sees a company at the center of a value network that includes its
suppliers, its immediate customers and their end customers. The value network includes
valued relations with others such as university researchers, government approval agencies
and so on.

PROCUREMENT & TRANSPORT

 Getting the raw material and packaging material requirement from the production unit
in charge.
 Constant updates on the procurement of materials and transport details.
 Production details and ingredient content information from the different personnel and
coordinating this activity.

PACKAGING

 Approval and coordination of the supply of packaging material to the production unit.

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CLEARING AND FORWARDING AGENTS (C&FA)

 From manufacturing plant the stock is transported or supplied to clearing and

forwarding agents.

 Clearing and Forwarding Agents is a third party and SPAN LABS Products gives
contract to them, so company has nothing to do in building the relationship with them.

 Here C&FA keep or stock the goods with them.

STOCKIEST OR DISTRIBUTORS

 Stockiest store the products in their godowns, C&FA supplies the goods to them
as per their order.
 Stockiest has some sales men working under him, they are known as stockiest
sales man. Their work is to place the products in the market and take order from
retailers and then supply goods to them.
 Sales man either take ready stock with them or they first take orders and then
supply goods later on.
 There is a beat which is a schedule route of sales man, means sales man has to
daily cover the route as mention in the beat.
 Merchandising, making products visible, pasting posters, putting banners, and
seeing that goods are properly placed in the retail outlets is also the duty of
stockiest sales man.
 Companies‘ sales officer keeps a check on the stockiest and monthly report is also
prepared which is further analyzed by ASM & ZSM.

RETAILERS

 Retailers are backbone of the company as they are the one who can take the
product on new heights or can bring it down to toes.
 Stockiest supplies goods to retailers and tries Persuading retailers to give the
brand special displays (using merchandising tools) to get affective brand presence,
and arranging it in more noticeable manner.

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 Classification of outlets in different type of markets is different according to their
sales volume.

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 AYURVEDA – An Overview
Ayurveda is an ancient science of life, a traditional and the oldest and most holistic medical
system available on the planet today. Its major premise involves the symbiosis of mind, body
and spirit. Any imbalance in this synthesis results in physical ailments. This ancient Indian
medicine seeks to reestablish the harmony between the mind, body and its environment. It
was placed in written form over 5,000 years ago in India, and was said to be a world
medicine dealing with both body and the spirit. Before the advent of writing, the ancient
wisdom of this healing system was a part of the spiritual oral tradition of the Vedic tradition.
This has been handed down to us by means of ancient venerable scripts as palm leaf books,
leather leaves, etc. The oldest works in Ayurveda still available are the Charaka Samhita,
Sushuta Samhita and Ashtanga Samgraha, among others.

"Ayu" means life and "Veda" means knowledge from the Vedic texts. This holistic science is
the knowledge of complete balance of the Body, Mind and Spirit, including emotions and
psychology, on all levels. Ayurveda includes in its consideration, longevity, rejuvenation and
self-realization therapies through herbs, diet, exercise, yoga, aromas, tantras, mantras, and
meditation.

It is said to have originated from Lord Brahma (Creator of the Universe, according to Indian
mythology) and descended to the earth through various generations of gods and saints. The
sage-physician-surgeons of the time were the same sages or seers, deeply devoted holy
people, who saw health as an integral part of spiritual life. It is said that they received their
training of Ayurveda through direct cognition during meditation. In other words, the
knowledge of the use of various methods of healing, prevention, longevity and surgery came
through Divine revelation (Cosmic Intelligence); there were no guessing or testing and
harming animals. These revelations were transcribed from the oral tradition into book form,
interspersed with the other aspects of life and spirituality.

The original ancient Ayurvedic scholars also comprehended a true method to study and fully
understand Ayurveda. The Vedic Way to study and understand Ayurveda is the same Vedic
Way one takes to study life itself.

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The Vedas describe life as a series of experiences. Each experience can be seen as the
fundamental unit of life. It may be overwhelming to try to understand life as a whole, but by
understanding each experience, one can, therefore, understand life itself.

Every experience has three components: (1) an experiencer, (2) an object of experience, and
(3) the experience itself.
 The experiencer is you.
 The object of the experience can be any stimulus or environmental influence, e.g. a
sight, a sound, a thought, a material object, a circumstance, a situation, a person, etc.
 The experience is the interaction between the experiencer and the object.

An experience takes place only with all three components.

The Vedic approach to understand each experience is threefold:

 Sravanam (receiving Truth),
 Mananam (contemplating Truth), and
 Nidhidhyasanam (absorbing Truth)

These are the three aspects of the study and understanding Ayurveda. It is from this approach

that Ayurveda derives its name, for it is a true path for learning the meaning of life. Every

popular book written by the burgeoning numbers of uninitiated Ayurvedic ‗authors‘ translates

the term Ayurveda as ―knowledge or science (ved) of life (ayu).‖ However it is clear that

very few individuals realize the profound meaning of this appellation.

Sravanam can be achieved through exposure to the primary Ayurvedic scriptures or other

numinous literature and through the teachings of knowledgeable professors.

Mananam can be individual or through discussion and debate with other students of

Ayurveda.
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Nidhidhyasanam can be approached through a specific form meditation, sometimes referred

to as reflection, which allows the truthful understanding to become absorbed Only by

absorbing Truth, can one truly understand these principles and put them to use.

It is not necessary to approach sravanam, mananam, and nidhidhyasanam in a serial order

(i.e. one after the other), but rather one can incorporate different levels of each throughout

one's study of Ayurveda. We can constantly aspire to incorporate all three in our studies and

in our lives and perhaps glimpse the same truths as did the ancient Vedic seers.

Ayurveda is a holistic system of healing which evolved among the Brahmin sages of ancient

India some 3000-5000 years ago. There are several aspects of this system of medicine which

distinguish it from other approaches to health care:

1. It focuses on establishing and maintaining balance of the life energies within us,
rather than focusing on individual symptoms.
2. It recognizes the unique constitutional differences of all individuals and therefore
recommends different regimens for different types of people. Although two people
may appear to have the same outward symptoms, their energetic constitutions may be
very different and therefore call for very different remedies.
3. Ayurveda is a complete medical system which recognizes that ultimately all
intelligence and wisdom flows from one Absolute source (Paramatman). Health
manifests by the grace of the Absolute acting through the laws of Nature (Prakriti).
Ayurveda assists Nature by promoting harmony between the individual and Nature by
living a life of balance according to her laws.
4. Ayurveda describes three fundamental universal energies which regulate all natural
processes on both the macrocosmic and microcosmic levels. That is, the same
energies which produce effects in the various galaxies and star systems are operating
at the level of the human physiology--in your own physiology. These three universal
energies are known as the Tridosha.
5. Finally, the ancient Ayurvedic physicians realized the need for preserving the alliance
of the mind and body and offers mankind tools for remembering and nurturing the

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 subtler aspects of our humanity. Ayurveda seeks to heal the fragmentation and
disorder of the mind-body complex and restore wholeness and harmony to all people.

HISTORY OF AYURVEDA

To see the world from which Ayurveda developed it is necessary to go back 4000 years.
Ayurveda had not yet been established. People lived close to the cycles of nature in a thriving
agrarian society on the banks of the River Indus, dependent on the abundance of the harvest
and the bounty of water for its survival. It was also a world in which the people were
subjected to the full force of the power of nature; torrents of rain and the fierce heat of the
sun, as well as the reassurance of spring returning and the joy of reaping a mature harvest.
Subservience to the power that controls these natural extremes was at the centre of everyday
life in a religious world full of rituals. Regular fire sacrifices were carried out to supplicate
the deities upon whose favour the world depended. Ritual performance was as central to
maintaining health as eating enough food; both were needed to live and flourish. To treat
disease, herbs and potions were used alongside the incantations of the priests. In fact the
priests were both doctors and religious specialists. Disease spread fast in these warm and
humid climes. Fear of illness and of the death of loved ones was an everyday reality.
According to their belief system disease could be imposed from the spiritual world, from an
accident, or from the natural world. Here is the world in which the eternal tradition and the
empirical experience of everyday life could meet and intermingle. It was out of such a
cultural context that Ayurveda developed. Here was a fast-changing society that was
exploring its ideals of religion, royalty, leadership, law, medicine, and family. Philosophical
insight expanded as agrarian culture flourished.

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22
 SCOPE OF AYURVEDIC MEDICINE
Classically, Ayurvedic Medicine was conceptualized and practiced as eight major clinical

subspecialties of medicine in addition to numerous adjunctive specialties. The eight major

subspecialties continue to be taught today and they include:

1. Internal Medicine (Kayachikitsa)

2. General Surgery (Shalya Tantra)

3. Otorhinolaryngology (Shalakya)

4. Pediatrics and Obstetric/Gynecology (Kaumarabhrtya)

5. Psychiatry (Bhutavidya)

6. Toxicology (Agada Tantra)

7. Nutrition, Detoxification and Rejuvenation (Rasayana Tantra)

8. Fertility and Virility (Vajikarana)

For every disease, there is information about: definition, etiology, prodrome, clinical
symptoms, pathophysiology, and prognosis, principles of treatment, medicines, diet, lifestyle
recommendations, and even etymology. This approach is strikingly similar to that of modern
medicine and even more comprehensive.
Over the last century, Ayurvedic Medicine has experienced a rebirth and has continued to
evolve its holistic approach to health in accordance with modern needs and scientific
advances of the day. Today, modern Ayurveda also includes:
1. Kulam Svastyam Kutumbakam: Principles of Preventative Healthcare For the
Entire Family
2. Sangakara Chikitsa: Treatment of Addictions. Includes strategies for defeating
addictions to alcohol, tobacco, sexual behavior, and food.
3. Panchakarma Chikitsa: Purification and Rejuvenation Treatments. Prescribed with
respect to one's individual nature, work, social circumstance, age, and season.
4. Sthaulya Chikitsa: The Ayurvedic Approach To Diet and Weight Loss. This
discipline covers practical and effective approaches to maintain a healthy weight
through constitutionally-determined diet, exercise, herbs, spices, teas, breathing, and
psychological aids.
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 5. Vatavyadhi Chikitsa: Specific treatment plans for the diseases of Vata origin which
affect the musculoskeletal system and nervous system (joints, bones, muscles, nerves)
Examples include but are not limited to: osteoarthritis, osteoporosis, osteopenia,
multiple sclerosis, spondylosis, sciatica, fibromyalgia, and chronic fatigue syndrome.
6. Svabhaavoparamavaada: Promotion of self-healing and resistance to disease (i.e.
immunity) as per your age, sex, occupation, nature, daily routine, medical history,
mental status, season, and region.
7. Vajikarana: Specific remedies for Male infertility and impotence as well as Female
infertility.
8. Saundarya Sadhana: Beauty and cosmetic treatments for men and women, including
skin, hair, eyes, posture, body odor, and general appearance.

BASIC PRINCIPLES OF AYURVEDA

The Three Doshas

The Five Elements
Everything in the universe is made up of combinations of the Five Elements (Pancha
Mahabhutas). This includes the human being which also acquires a soul or spirit. These five
elements are known as:
 Space or Akasha
 Air or Vayu
 Fire or Tejas
 Water or Apa
 Earth or Prithvi

These five elements, it should be understood, derive from and are expressions of an
unmanifest and undifferentiated Creative Principle, which is one. These five elements are to
be understood in a material sense as well as a subtle sense. By earth we are to understand not
only the terrain of our planet or the iron in our red blood cells and spleen, but also the quality
of steadfastness of mind, strength of one‘s moral fiber, one‘s slow and quiet undeterred
advancement towards a goal, and the resistance to the manifestations of others. By water we
mean to imply the cohesive aspects of reality which flows into and holds things together,
perfectly and simply witnessed in the ubiquitous H20 molecule. And the other elements too

24
were intended by the ancient vaidyas (physicians) to communicate the essential universal
principle inherent in a particular element. By fire we mean the universal force in nature that
produces heat and radiates light; it is our passion to pursue despite obstacles and delays; it is
what burns away the cloak of ignorance (avidya) and allows the Truth to shine with
brilliance. Fire removes doubt from the mother-substance of human heart and replaces it with
joy.
Air is that transparent, rarefied, kinetic force which sets the universe in motion; it moves the
blood through the vessels, wastes from the body, thoughts through the mind; it moves the
birds to warmer climates in winter, it moves the planets around their suns. Space is the
subtlest of all elements which is everywhere and touches everything; in the mind it is the
vessel which receives all impressions, in the heart space accepts love; space is receptivity and
non-resistance to what is true.

Thus these Five Subtle Elements (Pancha Mahabhutas) form the basis for all things found in
the material creation, from a grain of sand to the complex physiology of every human being.
Balancing these elements in just the right way for each unique individual is the key to
maintaining health and treating disease should it arise, whether it is physical, mental, or
spiritual.

The Tridosha

The five elements can be seen to exist in the material universe at all scales both organic and
inorganic, from peas to planets. When they enter into the biology of a living organism, man
for example, they acquire a biological form. This means that the five elements are coded into
three biological forces which govern all life processes. These three forces are known as the
three doshas, or simply thetridosha.. The tridosha regulates every physiological and
psychological process in the living organism. The interplay among them determines the
qualities and conditions of the individual. A harmonious state of the three doshas creates
balance and health; an imbalance, which might be an excess (vrddhi) or deficiency (ksaya),
manifests as a sign or symptom of disease.

The three doshas are known as Vata, Pitta, and Kapha.

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You can think of these three doshas as fundamental biological energies which regulate all the
life processes of an individual. And as we will discuss later, although all individuals are made
up of these same three energies, we all have them in unique proportions. The doshas obtain
their qualities by virtue of their elemental composition as we can see in the simple diagram
below. Each of the three doshas is composed of two elements as shown here:

Elements Composing the Tridosha

 Vata
Space (Akasha)
Air (Vayu)
 Pitta
Fire (Tejas)
Water (Apa)
 Kapha
Water (Apa)
Earth (Prithvi)

Thus, Vata is composed of space and air, Pitta of fire and water, and Kapha of water and
earth.. Vata dosha has the mobility and quickness of space and air; Pitta dosha the metabolic
qualities of fire and water; Kapha dosha the stability and solidity of water and earth.
Interestingly, the Sanskrit entomology of the word dosha gives it the meaning of "blemish,
that which darkens". This alerts us to the fact that when in balance these force are life-
supporting but when imbalanced they are the agents of disease and misery.

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 HOW THE AYURVEDIC TRADITION BECAME A SYSTEM OF
EMPIRICAL MEDICINE
Any history of ayurvedic development requires discussing two different perspectives; a linear
religio-historical approach and a circular organic expansion. The first perceives Ayurveda as
a timeless system of medicine where its knowledge is perfect and divinely inspired; the
second view is that ayurvedic medical knowledge has developed out of ritualistic healing into
an empirical medicine system that is grounded in clinical experience. The introductory verses
of ayurvedic texts reflect the perspective that Ayurveda is an eternal revelation. They all start
with a mythological account of the gods passing ayurvedic knowledge down to humans. This
divine stamp is a well-known Indian method of authenticating a text and making it orthodox
(Wujastyk 2003). It is a way of bringing formerly untraditional and perhaps unaccepted ideas
into mainstream culture. Much of the secondary and modern ayurvedic literature also implies
a consistent tradition that is divinely inspired and eternal.
But, as you untangle the web of influences that have affected Ayurveda the evidence clearly
reveals an expanding tradition that has accumulated knowledge over time and through
experience. This latter organic perspective, first introduced by Jan Meulenbeld, holds that
Ayurveda is a science of unfolding truth and as a path of discovery it has not and will not
remain static. These developments are not necessarily mutually exclusive, but it is useful to
understand the roots of different ayurvedic traits.
The concept of a timeless tradition has great appeal, for the insights of Ayurveda are
incredible and they do appear to be divinely inspired. How else have we learnt about the
properties of so many herbs and minerals? How was it discovered, for example, that brahmi
(Bacopa monniera) is so effective at improving the intellect and guggulu (Commiphora
mukul) so useful at reducing tumours? How did the pioneers of Ayurveda learn to diagnose
illness with only the five senses at their disposal? Having said this, the idea of human
knowledge growing through experience, logic and insight has great value. Human
development is firmly grounded in endeavour. For Indian minds this duality causes no
conflict as Ayurveda can be two things at the same time: both divinely inspired and open to
human adaptation. This is a powerful medium for expression of the truth as it is both
reductionist and holistic.

Taking the first paradigm, while there is nothing inherently wrong with the claim of eternal
divine origins there are some potential problems with this perspective. It could potentially
27
stifle new ideas within Ayurveda as, in order to gain validity, there is a tendency for clinical
experience to be referenced back to a divine eternal source. Humble that this approach is,
new ideas are not easily propagated. There is an element of this attitude displayed by the core
theoretical ground of Ayurveda, having remained very similar over the last 2000 years. The
relative lack of modern innovative ayurvedic literature generating improved methods of
treatment, in comparison to Chinese and Western herbal medicine, is perhaps partially a
result of this. It may be that the inherent theories of Ayurveda are already complete, but
effective clinical insights are always of benefit as new diseases and cultural habits arise.The
insistence on divine origins has stagnated this process of valuing both clinical experience and
theory. It is not therefore surprising that as Ayurveda has been under continual threat from
certain Moghul, British and, currently, allopathic forces in the last 400 years, it has in some
quarters been necessary to fall back on its ancient roots in order to validate and justify its
presence. This has protected but also weakened Ayurveda. Its strength is really in its present
clinical excellence and the ayurvedic community should be harnessing powerful social forces
and speaking with confidence about its ability to help our society. However, this is made
difficult when Ayurveda is presently only recognised as an adjunctive medical system, where
ayurvedic doctors can only hold the position of a third medical officer at primary health
centres in India, and complementary medicine the world over holds a similarly lowly position
in the medical hierarchy. As a literature base of over 2000 years, hundreds of thousands of
expert physicians, millions of healed patients and numerous positive clinical trials attest,
ayurvedic treatment works and practitioners and professional registers should promote this,
researchers should publish clinical data and governments should support it enthusiastically.
Although Ayurveda has its roots in the past, its practitioners must embrace the present.
Ayurveda and ayurvedic physicians deserve greater recognition than they receive today.
Another, and potentially more serious, problem of relying on a doctrine that holds its origins
as divinely and infallibly inspired, is that it can and has resulted in right-wing fundamental
political groups utilising it to their own ends. This is clearly the case in India today with the
current rise in popularity of right-wing fundamental Hindu groups, and shows how the
struggle for political supremacy can infect religion (and vice versa).

This insistence of the divine origins of Ayurveda may unwittingly reinforce this political
doctrine if it continues to ignore modern Indological historical knowledge. By this, it is
referred to certain quarters of the academic community promoting this ideology as though
28
Vedic knowledge has remained eternally and statically predominant in all aspects of Indian
culture for all time. The point is that while religion, medicine and politics are interrelated, the
potential repercussions of an ideology must be considered; in this case, pandering to extreme
political causes that oppose the central tenet of Ayurveda—caring for all humanity.

The second paradigm, the scientific dependence on empirical evidence, can also be taken too
far to the extreme, with similar detriment. This has occurred within the modern medical
paradigm of ‗evidencebased medicine‘ requiring ethically dubious double-blind clinical trials
and animal experiments with a heavy dependence on single active ingredients, synthesised
medicines, separate chemical pathways and a reductionist methodology that has lost the
holistic view.

Holding onto the primacy of either of these two paradigms means that the complete picture is
missed. As we shall see, Ayurveda can offer a balance to these extremes as it contains both
paradigms within it. I think this inner debate between tradition and progression is mirrored in
our everyday lives and specifically experienced when using natural medicine. The question is
how to respect tradition while integrating personal experience. Internally it is a case of
communication between heart and head where intuition and intellect are both valid. As we
shall see, intuition and intellect are both essential for medicine to be, as Ayurveda is, truly
holistic.

29
 QUALITY CONTROL OF AYURVEDIC DRUGS
Quality control for efficacy and safety of ayurvedic products is of paramount importance.
Quality can be defined as the status of a drug that is determined by identity, purity, content,
and other chemical, physical, or biological properties, or by the manufacturing processes.
Quality control is a term that refers to processes involved in maintaining the quality and
validity of a manufactured product. For the quality control of a traditional medicine, the
traditional methods are procured and studied, and documents and the traditional information
about the identity and quality assessment are interpreted in terms of modern assessment. In
general, all medicines, whether they are of synthetic or of plant origin, should fulfill the basic
requirements of being efficacious and safe, and this can be achieved by suitable clinical trials.
This applies both to the multinational pharmaceutical company conducting a multi-center,
double-blind placebo-controlled study with a herbal extract, and to the health practitioner in a
rural village who applies a locally produced ayurvedic mixture. Natural products in medicine
constitute a vast array of ―raw materials,‖ making clear definitions important. Quality criteria
are based on clear scientific definitions of the raw material. The term ―ayurvedic drugs‖
denotes plants or plant parts that have been converted into phytopharmaceuticals by means of
simple processes involving harvesting, drying, and storage. Hence they are capable of
variation. This variability is also caused by differences in growth, geographical location, and
time of harvesting. A practical addition to the definition is also to include other crude
products derived from plants, which no longer show any organic structure, such as essential
oils, fatty oils, resins, and gums. Derived or isolated compounds in the processed state such
as extracts or even isolated purified compounds (e.g. strychnine from Strychnos nux-vomica)
or mixtures of compounds (e.g. abrin from Abrus precatorius) are, as a rule, not included in
the definition. Combinations with chemically defined active substances or isolated
constituents, and homeopathic preparations which frequently contain plants, are not regarded
as ayurvedic medicines. Their production is already based on adequate quality control of the
respective starting materials. The following paragraphs will focus on quality control of
ayurvedic drugs in compliance with the above definition.

In general, quality control is based on three important pharmacopeial definitions:

 Identity: Is the herb the one it should be?

30
  Purity: Are there contaminants, e.g., in the form of other herbs which should not be
there?
 Content or assay: Is the content of active constituents within the defined limits?
It is obvious that the content is the most difficult one to assess, since in most ayurvedic drugs
the active constituents are unknown. Sometimes markers can be used which are, by
definition, chemically defined constituents that are of interest for control purposes,
independent of whether they have any therapeutic activity or not. To prove identity and
purity, criteria such as type of preparation sensory properties, physical constants,
adulteration, contaminants, moisture, ash content and solvent residues have to be checked.
The correct identity of the crude herbal material, or the botanical quality, is of prime
importance in establishing the quality control of ayurvedic drugs. Identity can be achieved by
macro- and microscopical examinations. Voucher specimens are reliable reference sources.
Outbreaks of diseases among plants may result in changes to the physical appearance of the
plant and lead to incorrect identification. At times an incorrect botanical quality with respect
to the labeling can be a problem. For example, in the 1990s, a South American product
labeled as ―Paraguay Tea‖ was associated with an outbreak of ant cholinergic poisoning in
New York. Subsequent chemical analysis revealed the presence of a class of constituents that
was different from the metabolites normally found in the plant from which Paraguay tea is
made.

Purity is closely linked with the safe use of drugs and deals with factors such ash values,
contaminants (e.g. foreign matter in the form of other herbs), and heavy metals. However,
due to the application of improved analytical methods, modern purity evaluation also
includes microbial contamination, aflatoxins, radioactivity, and pesticide residues. Analytical
methods such as photometric analysis, thin layer chromatography (TLC), high performance
liquid chromatography (HPLC), and gas chromatography (GC) can be employed in order to
establish the constant composition of herbal preparations. Depending upon whether the active
principles of the preparation are known or unknown, different concepts such as
―normalization versus standardization‖ have to be applied in order to establish relevant
criteria for uniformity.

Content or assay is the most difficult area of quality control to perform, since in most
ayurvedic drugs the active constituents are not known. Sometimes markers can be used. In all
31
other cases, where no active constituent or marker can be defined for the ayurvedic drug, the
percentage extractable matter with a solvent may be used as a form of assay, an approach
often seen in pharmacopeias. The choice of the extracting solvent depends on the nature of
the compounds involved, and might be deduced from the traditional uses. For example, when
an ayurvedic drug is used to make a tea, the hot water extractable matter, expressed as
milligrams per gram of air-dried material, may serve this purpose.
A special form of assay is the determination of essential oils by steam distillation. When the
active constituents (e.g. sennosides in Senna) or markers (e.g. alkydamides in Echinacea) are
known, a vast array of modern chemical analytical methods such as ultraviolet/visible
spectroscopy (UV/VIS), TLC, HPLC, GC, mass spectrometry (MS), or a combination of GC
and MS (GC/MS), can be employed.
Several problems not applicable to synthetic drugs influence the quality of ayurvedic drugs:
 Ayurvedic drugs are usually mixtures of many constituents.
 The active principle(s) is (are), in most cases unknown.
 Selective analytical methods or reference compounds may not be available
commercially.
 Plant materials are chemically and naturally variable.
 Chemo-varieties and chemo cultivars exist.
 The source and quality of the raw material are variable.
 The methods of harvesting, drying, storage, transportation, and processing (for
example, mode of extraction and polarity of the extracting solvent, instability of
constituents, etc.) have an effect.
Strict guidelines have to be followed for the successful production of a quality ayurvedic
drug. Among them are proper botanical identification, phytochemical screening, and
standardization. Quality control and the standardization of ayurvedic medicines involves
several steps. The source and quality of raw materials, good agricultural practices and
manufacturing processes are certainly essential steps for the quality control of ayurvedic
medicines and play a pivotal role in guaranteeing the quality and stability of ayurvedic
preparations.

The quality of a plant product is determined by the prevailing conditions during growth, and
accepted Good Agricultural Practices (GAP) can control this. These include seed selection,
growth conditions, and use of fertilizers, harvesting, drying and storage. In fact, GAP
32
procedures are, and will be, an integral part of quality control. Factors such as the use of fresh
plants, age and part of plant collected, period, time and method of collection, temperature of
processing, exposure to light, availability of water, nutrients, drying, packing, transportation
of raw material and storage, can greatly affect the quality, and hence the therapeutic value of
ayurvedic medicines.

Apart from these criteria, factors such as the method of extraction, contamination with
microorganisms, heavy metals, and pesticides can alter the quality, safety, and efficacy of
ayurvedic drugs. Using cultivated plants under controlled conditions instead of those
collected from the wild can minimize most of these factors.

Sometimes the active principles are destroyed by enzymic processes that continue for long
periods from collection to marketing, resulting in a variation of composition. Thus proper
standardization and quality control of both the raw material and the ayurvedic preparations
should be conducted. Standardization involves adjusting the ayurvedic drug preparation to a
defined content of a constituent or a group of substances with known therapeutic activity by
adding excipients or by mixing ayurvedic drugs or herbal drug preparations. Botanical
extracts made directly from crude plant material show substantial variation in composition,
quality, and therapeutic effects. Standardized extracts are high-quality extracts containing
consistent levels of specified compounds, and they are subjected to rigorous quality controls
during all phases of the growing, harvesting, and manufacturing processes. No regulatory
definition exists for standardization of dietary supplements. As a result, the term
―standardization‖ may mean many different things. Some manufacturers use the term
standardization incorrectly to refer to uniform manufacturing practices; following a recipe is
not sufficient for a product to be called standardized. Therefore, the presence of the word
―standardized‖ on a supplement label does not necessarily indicate product quality. When the
active principles are unknown, marker substance(s) should be established for analytical
purposes and standardization. Marker substances are chemically defined constituents of a
ayurvedic drug that are important for the quality of the finished product. Ideally, the chemical
markers chosen would also be the compounds that are responsible for the botanical‘s effects
in the body.

33
There are two types of standardization. In the first category, ―true‖ standardization, a definite
phytochemical or group of constituents is known to have activity. Ginkgo with its 26%
ginkgo flavones and 6% terpenes is a classic example. These products are highly
concentrated and no longer represent the whole herb, and are now considered as
phytopharmaceuticals. In many cases they are vastly more effective than the whole herb.
However the process may result in the loss of efficacy and the potential for adverse effects
and herb–drug interactions may increase. The other type of standardization is based on
manufacturers guaranteeing the presence of a certain percentage of marker compounds; these
are not indicators of therapeutic activity or quality of the herb. In the case of ayurvedic drug
preparations, the production and primary processing of the medicinal plant or herbal drug has
a direct influence on the quality of the active pharmaceutical ingredients (APIs). Due to the
inherent complexity of naturally growing medicinal plants and the limited availability of
simple analytical techniques to identify and characterize the active constituents solely by
chemical or biological means, there is a need for an adequate quality assurance system. This
assurance is also required during cultivation, harvesting, primary processing, handling,
storage, packaging, and distribution. Deterioration and contamination through adulteration,
especially microbial contamination, can occur at any one of these stages. It is extremely
important to establish good agricultural, harvesting, and manufacturing practices for
ayurvedic starting materials in order to minimize these undesirable factors.

In this regard producers, processors, and traders of medicinal plants or ayurvedic drugs have
an obligation and a role to play. The manufacturers and suppliers of ayurvedic products
should adhere to quality control standards and good manufacturing practices. Currently, only
a few manufacturers adhere to complete quality control and good manufacturing procedures
including microscopic, physical, chemical, and biological analysis. Organizations such as
Department of Ayush help safeguard our health by carrying out premarket reviews of all
drugs before they are authorized for sale. The products available in the market are analyzed
regularly to ensure that they are free of unsafe ingredients and that the products actually
contain the ingredients indicated on the labels.

The potency and quality of an individual ayurvedic product may be unclear because of lack
of regulation. It is obvious that for a given plant product its quality will also be determined by
the prevailing conditions during the growth cycle of the plant. Therefore, for cultivated plants
34
the GAP system has been introduced, under which each step, including seed selection,
growing conditions, use of fertilizers, and optimization of harvest time, harvesting, and
drying, has to adhere to a set of criteria. It is likely that GAP procedures will become an
integral part of quality control in the near future.

Microscopic Evaluation

Quality control of ayurvedic drugs has traditionally been based on appearance and today
microscopic evaluation is indispensable in the initial identification of herbs, as well as in
identifying small fragments of crude or powdered herbs, and detection of foreign matter and
adulterants. A primary visual evaluation, which seldom needs more than a simple magnifying
lens, can be used to ensure that the plant is of the required species, and that the right part of
the plant is being used. At other times, microscopic analysis is needed to determine the
correct species and/or that the correct part of the species is present. For instance, pollen
morphology may be used in the case of flowers to identify the species, and the presence of
certain microscopic structures such as leaf stomata can be used to identify the plant part used.
Although this may seem obvious, it is of prime importance, especially when different parts of
the same plant are to be used for different treatments. Stinging nettle (Urtica urens) is a
classic example where the aerial parts are used to treat rheumatism, while the roots are
applied for benign prostate hyperplasia.

Determination of Foreign Matter

Ayurvedic drugs should be made from the stated part of the plant and be devoid of other parts
of the same plant or other plants. They should be entirely free from moulds or insects,
including excreta and visible contaminant such as sand and stones, poisonous and harmful
foreign matter and chemical residues. Animal matters such as insects and ―invisible‖
microbial contaminants, which can produce toxins, are also among the potential contaminants
of ayurvedic medicines. Macroscopic examination can easily be employed to determine the
presence of foreign matter, although microscopy is indispensable in certain special cases (for
example, starch deliberately added to ―dilute‖ the plant material). Furthermore, when foreign
matter consists, for example, of a chemical residue, TLC is often needed to detect the
contaminants.

35
Determination of Ash

To determine ash content the plant material is burnt and the residual ash is measured as total
and acid-insoluble ash. Total ash is the measure of the total amount of material left after
burning and includes ash derived from the part of the plant itself and acid-insoluble ash. The
latter is the residue obtained after boiling the total ash with dilute hydrochloric acid, and
burning the remaining insoluble matter. The second procedure measures the amount of silica
present, especially in the form of sand and siliceous earth.

Determination of Heavy Metals

Contamination by toxic metals can either be accidental or intentional. Contamination by

heavy metals such as mercury, lead, copper, cadmium, and arsenic in ayurvedic remedies can
be attributed to many causes, including environmental pollution, and can pose clinically
relevant dangers for the health of the user and should therefore be limited. The potential
intake of the toxic metal can be estimated on the basis of the level of its presence in the
product and the recommended or estimated dosage of the product. This potential exposure
can then be put into a toxicological perspective by comparison with the so-called Provisional
Tolerable Weekly Intake values (PTWI) for toxic metals, which have been established by the
Food and Agriculture Organization of the World Health Organization (FAO-WHO).

A simple, straightforward determination of heavy metals can be found in many

pharmacopeias and is based on color reactions with special reagents such as thioacetamide or
diethyldithiocarbamate, and the amount present is estimated by comparison with a standard.
Instrumental analyses have to be employed when the metals are present in trace quantities, in
admixture, or when the analyses have to be quantitative. The main methods commonly used
are atomic absorption spectrophotometry (AAS), inductively coupled plasma (ICP) and
neutron activation analysis (NAA).

Determination of Microbial Contaminants and Aflatoxins

Medicinal plants may be associated with a broad variety of microbial contaminants,

represented by bacteria, fungi, and viruses. Inevitably, this microbiological background

36
depends on several environmental factors and exerts an important impact on the overall
quality of ayurvedic products and preparations. Risk assessment of the microbial load of
medicinal plants has therefore become an important subject in the establishment of modern
Hazard Analysis and Critical Control Point (HACCP) schemes.

Ayurvedic drugs normally carry a number of bacteria and molds, often originating in the soil.
Poor methods of harvesting, cleaning, drying, handling, and storage may also cause
additional contamination, as may be the case with Escherichia coli or Salmonella spp. While
a large range of bacteria and fungi are from naturally occurring microflora, aerobic spore-
forming bacteria frequently predominate.

Laboratory procedures investigating microbial contaminations are laid down in the well-
known pharmacopeias, as well as in the WHO guidelines. Limit values can also be found in
the sources mentioned. In general, a complete procedure consists of determining the total
aerobic microbial count, the total fungal count, and the total Enterobacteriaceae count,
together with tests for the presence of Escherichia coli, Staphylococcus aureus, Shigella, and
Pseudomonas aeruginosa and Salmonella spp. The European Pharmacopoeia also specifies
that E. coli and Salmonella spp. should be absent from ayurvedic preparations. However it is
not always these two pathogenic bacteria that cause clinical problems. For example, a fatal
case of listeriosis was caused by contamination of alfalfa tablets with the Grampositive
bacillus Listeria monocytogenes.

Materials of vegetable origin tend to show much higher levels of microbial contamination
than synthetic products and the requirements for microbial contamination in the Indian
Pharmacopoeia allow higher levels of microbial contamination in ayurvedic remedies than in
synthetic pharmaceuticals. The allowed contamination level may also depend on the method
of processing of the drug. For example, higher contamination levels are permitted if the final
ayurvedic preparation involves boiling with water.

The presence of fungi should be carefully investigated and/or monitored, since some common
species produce toxins, especially aflotoxins. Aflatoxins in ayurvedic drugs can be dangerous
to health even if they are absorbed in minute amounts. Aflatoxin-producing fungi sometimes
build up during storage. Procedures for the determination of aflatoxin contamination in
37
ayurvedic drugs are published by the WHO. After a thorough clean-up procedure, TLC is
used for confirmation. In addition to the risk of bacterial and viral contamination, ayurvedic
remedies may also be contaminated with microbial toxins, and as such, bacterial endotoxins
and mycotoxins, at times may also be an issue. There is evidence that medicinal plants from
some countries may be contaminated with toxigenic fungi (Aspergillus, Fusarium). Certain
plant constituents are susceptible to chemical transformation by contaminating
microorganisms.

Withering leads to enhanced enzymic activity, transforming some the constituents to other
metabolites not initially found in the herb. These newly formed constituent(s) along with the
molds such as Penicillium nigricans and P. jensi may then have adverse effects.

Determination of Pesticide Residues

Even though there are no serious reports of toxicity due to the presence of pesticides and
fumigants, it is important that herbs and ayurvedic products are free of these chemicals or at
least are controlled for the absence of unsafe levels. ayurvedic drugs are liable to contain
pesticide residues, which accumulate from agricultural practices, such as spraying, treatment
of soils during cultivation, and administering of fumigants during storage. However, it may
be desirable to test ayurvedic drugs for broad groups in general, rather than for individual
pesticides. Many pesticides contain chlorine in the molecule, which, for example, can be
measured by analysis of total organic chlorine. In an analogous way, insecticides containing
phosphate can be detected by measuring total organic phosphorus.
Samples of ayurvedic material are extracted by a standard procedure, impurities are removed
by partition and/or adsorption, and individual pesticides are measured by GC, MS, or
GC/MS. Some simple procedures have been published by the Department of Ayush and the
Indian Pharamacopoeia has laid down general limits for pesticide residues in medicine.

Analytical Methods

Published monographs in a pharmacopeia are the most practical approach for quality control
of ayurvedic drugs and there are many available. When pharmacopeial monographs are
unavailable, development and validation of analytical procedures have to be carried out by
the manufacturer. The best strategy is to follow closely the pharmacopeial definitions of
38
identity, purity, and content or assay. Valuable sources for general analytical procedures are
included in the pharmacopeias, in guidelines published by the Department of Ayush .
Additional information, especially on chromatographic and/or spectroscopic methods can be
found in the general scientific literature. The plant or plant extract can be evaluated by
various biological methods to determine pharmacological activity, potency and toxicity. A
simple chromatographic technique such as TLC may provide valuable additional information
to establish the identity of the plant material. This is especially important for those species
that contain different active constituents.

Qualitative and quantitative information can be gathered concerning the presence or absence
of metabolites or breakdown products. TLC fingerprinting is of key importance for ayurvedic
drugs made up of essential oils, resins, and gums, which are complex mixtures of constituents
that no longer have any organic structure. It is a powerful and relatively rapid solution to
distinguish between chemical classes, where macroscopy and microscopy will fail.

Chromatograms of essential oils, for example, are widely published in the scientific literature,
and can be of invaluable help in identification.

The instruments for UV-VIS determinations are easy to operate, and validation procedures
are straightforward but at the same time precise. Although measurements are made rapidly,
sample preparation can be time consuming and works well only for less complex samples,
and those compounds with absorbance in the UV-VIS region.

HPLC is the preferred method for quantitative analysis of more complex mixtures. Though
the separation of volatile components such as essential and fatty oils can be achieved with
HPLC, it is best performed by GC or GC/MS.

The quantitative determination of constituents has been made easy by recent developments in
analytical instrumentation. Recent advances in the isolation, purification, and structure
elucidation of naturally occurring metabolites have made it possible to establish appropriate
strategies for the determination and analysis of quality and the process of standardization of
ayurvedic preparations. Classification of plants and organisms by their chemical constituents
is referred to as chemotaxonomy. TLC, HPLC, GC, quantitative TLC (QTLC), and high-
39
performance TLC (HPTLC) can determine the homogeneity of a plant extract. Over-
pressured layer chromatography (OPLC), infrared and UV-VIS spectrometry, MS, GC, liquid
chromatography (LC) used alone, or in combinations such as GC/MS, LC/MS, and MS/MS,
and nuclear magnetic resonance (NMR), electrophoretic techniques, especially by
hyphenated chromatographies, are powerful tools, often used for standardization and to
control the quality of both the raw material and the finished product. The results from these
sophisticated techniques provide a chemical fingerprint as to the nature of chemicals or
impurities present in the plant or extract.

Based on the concept of photoequivalence, the chromatographic fingerprints of ayurvedic

medicines can be used to address the issue of quality control. Methods based on information
theory, similarity estimation, chemical pattern recognition, spectral correlative
chromatograms (SCC), multivariate resolution, the combination of chromatographic
fingerprints and chemometric evaluation for evaluating fingerprints are all powerful tools for
quality control of ayurvedic products.

Validation

The validation of ayurvedic products is a major public health concern both in developed and
resource-poor countries, where fake businesses selling adulterated ayurvedic medicines are
common. In this regard, there is no control by the government agencies, despite the existence
of certain guidelines in some individual countries and those outlined by the WHO. If the
ayurvedic products are marketed as therapeutic agents, and irrespective of whether the
products really have any positive effects to cure and reduce the severity of the disease, it is
necessary to ensure scientific validation and periodic monitoring of the quality and efficacy
by drug control administrators.

It is feasible that the introduction of scientific validation would control the production of
impure or adulterated ayurvedic products and would eventually ensure their rational use. This
could also lead to the regulation of the industry so that only qualified physicians and health
providers are allowed to prescribe the medication.

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Several of the principal pharmacopeias contain monographs outlining standards for ayurvedic
drugs. The major advantage of an official monograph published in a pharmacopeia is that
standards are defined and available, and that the analytical procedures used are fully
validated. This is of major importance, since validation can be a rather time-consuming
process.

By definition, validation is the process of proving that an analytical method is acceptable for
its intended purpose for pharmaceutical methods. Guidelines from the United States
Pharmacopeia (USPC, 1994–2001), the International Conferenceon Harmonization (ICH),
and the US Food and Drug Administration (FDA) provide a framework for performing such
validations. In general, validation investigations must include studies on specificity, linearity,
accuracy, precision, range, detection, and quantitative limits, depending on whether the
analytical method used is qualitative or quantitative. Also of utmost importance is the
availability of standards. For macroscopic and microscopic procedures in general this means
that reliable reference samples of the plant must be available. A defined botanical source (e.g.
voucher specimens) will normally solve this problem. Standards for chromatographic
procedures are less easy to obtain. Characteristic plant constituents, either active or markers,
are seldom available commercially. Sometimes an LC/MS approach can be referred to as a
mode of characterization. Going one step further, after isolation of such a compound,
elucidations to prove its definite structure will not be easy. The method often employed is to
use readily available compounds that behave similarly in the chosen chromatographic
systems, and to calculate retention values and/or times towards these compounds as a
standard.

Qualitative chemical examination is designed to detect and isolate the active ingredient(s).
TLC and HPLC are the main analytical techniques commonly used. In cases when active
ingredients are not known or too complex, the quality of plant extracts can be assessed by a
―fingerprint‖ chromatogram.

41
Ayurvedic Supplements

A botanical is a plant or part of a plant valued for its medicinal or therapeutic properties,
flavor, and/or scent. Herbs are subsets of botanicals. To be classified as a dietary supplement,
a botanical must meet the following criteria:
1. It is intended to supplement the diet.
2. It contains one or more dietary ingredients (including amino acids, vitamins, minerals,
herbs, or other botanicals, etc.).
3. It is intended to be taken orally as a pill, capsule, tablet, or liquid.
4. It is labeled as being a dietary supplement.

An ayurvedic supplement labeled ―Natural‖ does not mean it is safe or without any harmful
effects. Ayurvedic products can act the same way as drugs. Their safety depends on factors
such as their chemical make-up, how they work in the body, method of preparation, and
dosage. In the US, the FDA regulates herbal and other dietary supplements. This means that
they do not have to meet the same standards as drugs and over-the-counter medications, they
are not required to be standardized, and no legal or regulatory definitions exist for
standardization. As a result, manufacturers are not required to demonstrate the safety and
effectiveness of their products before they reach the market. In addition, they do not have to
adhere to any of the quality control measures applicable to drugs; hence the composition may
vary greatly from one batch to another. The use of some ayurvedic supplements has been
reported to be associated with ailments such as oral manifestations, including swelling,
irritation, and bleeding of the tongue. These potential effects of ayurvedic supplements, in
conjunction with factors related to regulation restrictions, suggest that the use of these
products may be associated with various adverse reactions that can affect health. The active
ingredient (s) in many ayurvedic supplements is not known and some have been found to be
contaminated with metals, unlabeled prescription drugs, and microorganisms. Under its
current regulatory authority, the Department of Ayush can remove a ayurvedic supplement
from the market only after it has been shown to be unsafe. There has been an increase in the
number of Internet websites that sell and promote ayurvedic supplements. Unfortunately,
some of them make inaccurate claims and statements regarding their products and claim
unsubstantiated effects in curing disease and disease conditions.

42
Adulteration of Ayurvedic drugs

Direct or intentional adulteration of drugs usually includes practices in which a ayurvedic

drug is substituted partially or fully with other inferior products. Due to morphological
resemblance to the authentic herb, many different inferior commercial varieties are used as
adulterants. These may or may not have any chemical or therapeutic potential. Substitution
by ―exhausted‖ drugs entails adulteration of the plant material with the same plant material
devoid of the active constituents.
This practice is most common in the case of volatile oil-containing materials, where the dried
exhausted material resembles the original drug but is free of the essential oils. Foreign matter
such as other parts of the same plant with no active ingredients, sand and stones,
manufactured artifacts, and synthetic inferior principles are used as substitutes. The practice
of intentional adulteration is mainly encouraged by traders who are reluctant to pay premium
prices for herbs of superior quality, and hence are inclined to purchase only the cheaper
products. This encourages producers and traders to sell herbs of inferior quality. Rarity of an
herbal product is another factor that influences adulteration. Sometimes sale of inferior
products may be unintentional.

In the absence of proper means of evaluation, an authentic drug partially or fully devoid of
the active ingredients may enter the market. Factors such as geographical sources, growing
conditions, processing, and storage are all factors that influence the quality of the drug.
Deterioration may contribute to indirect adulteration, and crude drugs are often prone to
deterioration, especially during storage, leading to the loss of the active ingredients,
production of metabolites with no activity and, in extreme cases, the production of toxic
metabolites. Physical factors such as air (oxygen), humidity, light, and temperature can bring
about deterioration directly or indirectly. These factors, alone or in combination, can lead to
the development of organisms such as molds, mites, and bacteria. Oxidation of the
constituents of a drug can be brought about by oxygen in the air, causing some products, such
as essential oils, to resinify or to become rancid. Moisture or humidity and elevated
temperatures can accelerate enzymatic activities, leading to changes in the physical
appearance and decomposition of the herb.

43
Dried herbs are particularly prone to contamination with spores of bacteria and fungi present
in the air. Bacterial growth is usually accompanied by the growth of molds, whose presence
is evidenced by changes in appearance; break down of the plant material, and smell. Mites,
nematode worms, insects/moths, and beetles can also destroy ayurvedic drugs during storage.

Control measures to protect against deterioration include the use of airtight containers made
of materials that will not interact physically or chemically with the material being stored.
Storage in ventilated, cool, dry areas and periodic spraying of the stored area with
insecticides will help to prevent the spread of infestation.

Sterilization of crude drugs is achieved by treatment of bulk consignments with ethylene

oxide, and methyl bromide under controlled conditions and complying with acceptable limits
for toxic residues. Markets from time to time experience wild fluctuations in the price of
herbals. One reason for this is indiscriminate harvesting which leads to the extinction of
natural populations – still the only source of bioresources. This in turn encourages producers
to replace the required herb with other supplements.

Contamination of Ayurvedic drugs and Herb–Drug Interactions

Conventional synthetic pharmaceuticals such as synthetic corticosteroids, nonsteroidal anti-

inflammatory drugs and other prescription drugs, potent drugs such as phenylbutazone, in
fact examples of almost every therapeutic drug class have been found in certain herbal
remedies as contaminants. A recent study by Ramsay et al. found that potent corticosteroids
had been deliberately added to ayurvedic creams in order increase their efficacy. This
problem is widespread, and occurs in both Asian and European countries. These
―adulterated‖ ayurvedic medicines sometimes result in serious ailments such as acute renal
failure.

Many people, especially those living with HIV/AIDS, use both ayurvedic medicines and
prescription drugs. A number of clinically significant interactions between prescribed and
ayurvedic medicines have been identified. When these medications are used together, they
can interact in the body, causing changes in the way the herbs and/or the drug works. Such
changes are called herb–drug interactions. Concurrent use of ayurvedic or homeopathic

44
remedies alongside prescribed or over-the-counter medicines are frequent, and may mimic,
magnify, or oppose the effect of the drug.

Herb–drug interactions are not chemical interactions between a drug and a ayurvedic
component to produce something toxic. Instead, the interactions generally cause either an
increase or decrease in the amount of drug in the bloodstream. As with conventional
medicines, ayurvedic medicines interact with drugs in two general ways: pharmacokinetically
and pharmacodynamically. Pharmacokinetic interactions result in alterations in the
absorption, distribution, metabolism, or elimination of the drug or natural medicine. These
interactions affect drug action by quantitative alterations, either increasing or decreasing the
amount of drug available to have an effect. Pharmacodynamic interactions cause alterations
in the way a drug or natural medicine affects a tissue or organ system. These actions affect
drug action in a qualitative way, either through enhancing or antagonizing effects.

Herb–drug interactions change the effectiveness of the treatment, sometimes resulting in

potentially dangerous side effects, possibly leading to toxicity, and/or reduced benefits. They
can modify the mode of action of the drug, leading to unexpected complications or
enhancement of the therapeutic effect, possibly leading to overmedication and an impact on
health. Drug interactions are a significant problem in association with the use of St John‘s
wort.

The risk of herb–drug interactions is not limited to synthetic drugs. Ayurvedic supplements
and certain foods can interact with medications. Unfortunately very little is known about
these interactions and there is little available scientific research on herb–drug interactions.
When combining ayurvedic therapies with other medications, it is important to watch for
potential symptoms and to inform health care providers. It is essential to train doctors to
appreciate that drug interactions exist and to emphasize the importance of the need for
physicians and naturopathic doctors to work together.
Currently, there is very little information published on herb–drug interactions. Controlled
clinical studies are needed to clarify and determine their clinical importance and more
research is required to define them.

45
Toxicity of Ayurvedic drugs

For several reasons it is not possible to establish absolute safety standards for ayurvedic
preparations based solely on epidemiological studies. First, these types of studies would be
costly. Second, there is little published data in countries where the major use of medicinal
plants occurs and thus general standards based on a limited number of reports would have
little meaning. Third, the exact identification of the products implicated in side effects
claimed for medicinal plants is usually lacking.
In spite of these inadequacies, there are a number of general comments that can be made with
regard to avoiding potential serious side effects from ayurvedic medicines. The definition of
―toxic‖ is ultimately a matter of viewpoint. Traditionally, herbs and ayurvedic products have
been considered to be nontoxic and have been used by the general public and traditional
medicinal doctors worldwide to treat a range of ailments. The fact that something is natural
does not necessarily make it safe or effective. The active ingredients of plant extracts are
chemicals that are similar to those in purified medications, and they have the same potential
to cause serious adverse effects. Whilst the literature documents severe toxicity resulting
from the use of herbs, on many occasions the potential toxicity of herbs and ayurvedic
products has not been recognized. In India, herbs can be obtained from temples, night
markets, street vendors, herbal stores, neighborhoods, or relatives, and from traditional
medicine practitioners. Ordinary people recommend the medicines to others without safety
considerations. The general public and many practitioners also believe that the herbs are
nontoxic. Apparently, this cultural style/concept needs more attention in terms of drug safety
education. Herbs and ayurvedic preparations can cause toxic adverse effects, serious allergic
reactions, adverse drug interactions, and can interfere with laboratory tests. High-risk patients
such as the elderly, expectant mothers, children, those taking several medications for chronic
conditions, those with hypertension, depression, high cholesterol or congestive heart failure,
should be more cautious in taking ayurvedic medicine. It is axiomatic that pregnancy should
be a time of minimal medical intervention, and vaids in particular regard pregnancy as a
―contraindication‖ to taking ayurvedic medicines.

Two kinds of side effects have been reported for ayurvedic medicines. The first, considered
to be intrinsic to ayurvedic drugs themselves, is mainly related to predictable toxicity due to
toxic constituents of the ayurvedic ingredients and overdosage, and the second is allergy.
Many cases of allergic reactions have been reported for ayurvedic drugs. It is impossible to
46
completely eliminate the possibility of any substance, including prescription drugs, ayurvedic
remedies, or cosmetics, producing an allergic response in people exposed to them. Ayurvedic
medicines do not present any more of a problem in this respect than any other class of widely
used foods or drugs.
Based on published reports, the side effects or toxic reactions associated with ayurvedic
medicines in any form are rare. This could be due to the fact that ayurvedic medicines are
generally safe, that adverse reactions following their use are underreported, or because the
nature of the side effects or minor allergic reactions is such that they are not reported.

Perhaps the major problem with regard to the safety of ayurvedic medicines is related to the
manufacturing practice, including contamination, substitution, incorrect preparation and
dosage, intentional addition of unnatural toxic substances, interactions involving synthetic
prescriptions, drugs, and ayurvedic medicines, either intentional or unintentional mislabeling,
and the presence of natural toxic contaminants.

Many ordinary foods contain constituents that could be regarded as poisonous. Alpha gliadin
produced by gluten in wheat, oats, and rye, the cyanogenic glycosides in many fruit skins and
seeds, thiocyanates of the brassica vegetables, and lectins of many pulses including soya and
red kidney bean are such examples. Cyanogenetic glycodides present in the kernel of many
fruits can undergo gastric hydrolysis, resulting in the release of hydrogen cyanide.
Viscotoxins, which are constituents of mistletoe, are both cytoxic and cardiotoxic.
Nonetheless, these foods are generally regarded as safe. Similarly, both water and oxygen can
kill in excessive amounts! So quantity is often an important consideration.
A number of cases have been reported in the literature in which ayurvedic medicines, used
for a number of years with safety, suddenly appear to be unsafe, and to date there has been no
satisfactory explanation for these adverse effects. In this context herbs can be broadly
classified into three major categories:
 The food herbs – medicines such as peppermint, ginger, garlic, hawthorn, nettles,
lemon, and balm are gentle in action, have low toxicity, and are unlikely to cause any
adverse response. They can be consumed in substantial quantities over long periods of
time without any acute or chronic toxicity. However they may bring about allergic
reactions in certain individuals.

47
  The medicinal herbs – these are not daily ―tonics‖ and need to be used with greater
knowledge (dosage and rationale for use) for specific conditions (with a medical
diagnosis) and usually only for a limited period. They have a greater potential for
adverse reactions and in some cases drug interactions. They include aloe vera, black
cohosh, comfrey, echinacea, ephedra, ginkgo biloba, ginseng, kava kava, milk thistle,
and senna.
 The poisonous herbs have a strong potential for either acute or chronic toxicity and
should only be prescribed by trained clinicians who understand their toxicology and
appropriate use.

Fortunately, the vast majority of these herbs is not available to the public and is not sold in
health food or ayurvedic stores. Aconite, Arnica spp., Atropa belladonna, digitalis, datura,
male fern, gelsemium, and veratrum are some examples.

There are herbs such as Lobelia and Euonymus spp. that have powerful actions, often causing
nausea or vomiting, although they are safe under appropriate conditions. There is also an
idiosyncratic grouping of herbs that have been alleged, with some scientific support, to
exhibit specific kinds of toxicity. The best known example is the hepatotoxicity of
pyrrolizidine alkaloid-containing plants such as Symphytum (comfrey), Dryopteris (male
fern), Viscum (mistletoe), and Corynanthe (Yohimbe).

Screening of Ayurvedic drugs

Once the botanical identity of a herb is established, the next step is phytochemical creening,
which involves bioassays, extraction, purification, and characterization of the active
constituents of pharmaceutical importance. The herb or ayurvedic drug preparation in its
entirety is regarded as the active substance. These constituents are either of known
therapeutic activity or are chemically defined substances or a group of substances generally
accepted to contribute substantially to the therapeutic activity of a ayurvedic drug. In any
program in which the end product is to be a drug, some type of pharmacological screening, or
evaluation, must obviously be done.

48
Pharmacological screening programs are not without problems. Ideally the active principles
should be isolated, preferably using bioassay guided isolation processes, which can be
problematic. The ideal pharmacological screen would be to identify those extracts or pure
compounds that are highly active and nontoxic. Such a screen is rare to find. Failure to
duplicate pharmacological results is another problem. There are many pharmacological
screening tests available. In the random selection program of the National Cancer Institute
(NCI) in the US, plants are randomly selected, extracted, and the extracts are evaluated
against one or more in vitro tumor systems and in vitro cytotoxicity tests. An extension of
this procedure is to isolate metabolites or ―active compounds‖ from the plant that had shown
most promising activity and subject them to pharmacological tests. In another approach,
plants containing specific types or classes of chemical compounds, for example alkaloids, are
tested. Simple tests such as color reactions are carried out on various parts of the plant in the
field, and assays are carried out in the laboratories. In terms of cost–benefit ratio, these
―shotgun‖ approaches are considered to be very unsatisfactory.
Another method involves random collection of plants and subjection of their extracts to
several broad screening methods and pharmacological tests. The success of this method
depends on the number of samples assayed, adequate funding, and appropriate predictable
bioassay protocols. Broad-based empirical screening, which is time consuming and
expensive, can detect novel activities but is not suited for screening large numbers of
samples.

Diagnosis by observation, a method introduced by the ―father‖ of medicine, Hippocrates, is

still one of the most powerful tools of today‘s physicians. In vitro screening methods, though
restricted to the detection of defined activities, are simpler and more useful. Recently,
biochemical and receptor–ligand binding assays have gathered momentum. This has been
made possible by the increasing availability of human receptors from molecular cloning, and
extracts and compounds can be tested for binding directly to the presumed therapeutic target
protein. Clone receptors can be expressed in a functional state linked to receptor proteins in
cells such as yeast, and this has been made possible by applications of molecular biology.
Combined with automated instrumentation and computer databases, hundreds of such assays
can be completed in relatively short periods of time. These screening processes are
successfully used by international agencies such as the National Cancer Institute (NCI) in the
United States and the Central Drug Research Institute in India.
49
The technology of plant medicinal screening processes has even advanced to enzyme
isolation. The enzymes that cause the disease are first isolated and the plant extracts are tested
to determine if they block enzyme action. An enzyme immunoassay for the quantification of
femtomole quantities of therapeutically important alkaloids has been established. Ethanolic
extracts, tinctures, and pure plant compounds from commercially available herbs have been
analyzed for their in vitro cytochrome P450 3A4 (CYP3A4) inhibitory capability via a
fluorometric microtiter plate assay. These studies indicate that high-throughput screening
methods for assessing CYP3A4 inhibition by natural products have important implications
for predicting the likelihood of potential herb–drug interactions.

Higher plants contain both mutagens and antimutagens and are susceptible to mutagenesis,
but screening programs for the detection of antimutagenesis rarely employ higher plant
systems. However, using modified screening tests to detect antimutagenic agents, higher
plants have been shown to contain a variety of structurally novel antimutagenic agents. Short-
term bacterial and mammalian tissue culture systems are the standard methods employed.

Labeling of Ayurvedic Products

The quality of consumer information about the product is as important as the finished
Ayurvedic product. Warnings on the packet or label will help to reduce the risk of
inappropriate uses and adverse reactions. The primary source of information on ayurvedic
products is the product label. Currently, there is no organization or government body that
certifies an herb or a supplement as being labeled correctly.

It has been found that ayurvedic remedy labels often cannot be trusted to reveal what is in the
container. Studies of ayurvedic products have shown that consumers have less than a 50%
chance of actually getting what is listed on the label, and published analyses of ayurvedic
supplements have found significant differences between what is listed on the label and what
is in the bottle. The word ―standardized‖ on a product label is no guarantee of higher product
quality, since there is no legal definition of the word ―standardized.‖ Consumers are often left
on their own to decide what is safe and effective for them and the lack of consistent labeling
on ayurvedic products can be a source of consumer frustration.

50
Certain information such as ―the product has been manufactured according to Pharmacopoeia
standards,‖ listing of active ingredients and amounts, directions such as serving quantity
(dosage) and frequency of intake of the drug, must be included on the labels of all ayurvedic
products and packages. The label should also indicate the method of extraction and relative
amount of macerate and menstruum used, and possible side effects. It should indicate that the
product‘s content has been standardized to contain a particular amount of a specified
biochemical constituent. Standardization gives the buyers a measure of potency by which to
judge the quality of the product and to compare dosage with those indicated by clinical trials.
This will also ensure that the correct herb has been used. In addition to the above information,
the label should include the name and origin of the product, its intended use, net quantity of
contents, other ingredients such as herbs and amino acids, and additives, for which no daily
values have been established, storage conditions, shelf life or expiry date, warnings,
disclaimer, and name and address of manufacturer, packer or distributor. A herb categorized
as a nutritional supplement cannot claim any health benefits or ―disease claims‖ on the label,
leaving the consumer with little information.

Marketing plays a big role in the use of ayurvedic products and the media help significantly
to provide information about natural health products. One of the problems with mass media
―propaganda‖ is scientific inconsistency. Unless the packaging contains a medical claim,
ayurvedic products are not reviewed by any government agency. Food and drug
administrations that regulate prescription drugs only review a ayurvedic product if the item is
suspected of being harmful or if the label contains medical claims. Scientists use several
approaches to evaluate botanical dietary supplements for their potential health benefits and
safety risks, including their history of use and laboratory studies using cell or animal models.
Studies involving people can provide information that is relevant as to how botanical dietary
supplements are used.

Policies and Regulations

It is a widely held myth that modern drugs are dangerous foreign chemicals with side effects,
while ayurvedic medicines are natural, gentle and safe. The truth is that some herbs can be
dangerous and can bring about serious diseases and even lead to death. Unlike conventional

51
drugs, ayurvedic products are not regulated for purity and potency and this could cause
adverse effects and can even lead to drug interactions. There are fewer studies on ayurvedic
medicines than on conventional drugs, mainly because, unlike synthetic chemicals, herbs
cannot be patented, so there is little money to be made by funding such research. It is
important that consumers are made aware of interactions herbs might have with other drugs
they are taking. Unfortunately this information is not available with ayurvedic medicines.
Ayurvedic medicines are also frequently adulterated with prescription drugs. In certain
countries, ayurvedic products used for diagnosis, cure, mitigation, treatment, or prevention of
disease are normally treated as drugs, and hence regulated by legislation.

However, in most countries, including the United States, such legislation does not exist and
in fact, most botanical products are marketed as dietary supplements. Ayurvedic products
categorized as nutritional or dietary supplements are not regulated. In many countries these
medicines are not required to pass any regulatory analysis to be sold as health food
supplements. It is clear that the ayurvedic industry needs to follow strict guidelines and that
regulations are needed. The food and drug administration‘s that regulate prescription drugs
only review a ayurvedic product if the item is suspected of being harmful or if the label
contains a medical claim. Although research is being done, it is very limited and only a few
ayurvedic drugs have been studied adequately by well-controlled clinical trials. Even though
evidence should always be presented to support claims of products, most herbs are still
marketed with little or no research. To be registered as drugs, these products need to be tested
to prove their safety and clinical efficacy. However, so far, few programs have been
established to study the safety and efficacy of ayurvedic medicines as originally proposed in
the WHO guidelines for the assessment of herbal medicines.

The future of ayurvedic drugs is overshadowed by the pervading lack of regulatory control.
In 1993, the WHO sponsored a symposium on the use of medicinal plants. The result was a
standard guideline for the assessment of herbal medicines and a recommendation that
governments of the world should protect medicinal plants, improve regulation of herbal
medicines, and respect traditional medicine approaches.

More recently the Department of Ayush developed a new regulatory framework for natural
health products, which came into effect in 1995.
52
Among other things, the new regulations call for improved labeling, good manufacturing
practices, product and site licensing, and provision of a full range of health claims that will be
supported by evidence. However, even in India, the only regulatory requirements enforced
are that all products intended for medicinal use, including natural health products, are issued
a Drug Identification Number (DIN). These numbers are not required for raw materials such
as bulk herbs. In the US, access to herbal medicines is restricted by FDA regulations. Before
any new chemical or ayurvedic drug is approved, research must prove that it is both safe and
effective. As a result of these restrictions, packages of ayurvedic medicines are labeled as
food supplements, which do not require pre-approved testing. Food supplements cannot make
any healing claims or issue warnings about potential risks. In the US, plant-based derivatives
already appear in a quarter of the prescription medicines produced. However, many other
plants with healing properties are shunned by the medical community despite scientific data
from other countries showing their effectiveness. The misconception that herbs are old
fashioned and unscientific has helped to promote a general distrust of phytotherapy. The
Central Drug Research Institute contends that, in many cases, ayurvedic medicines are safer
than prescription drugs. According to the CDRI, ayurvedic medicines react more slowly and
often include their own antidotes to counteract any toxic effects.

With proper enforcement of regulations, more products that are legitimate will enter the
market and the consumers will see justifiable claims on labels. In fact, it is predicted that
appropriate regulations will rejuvenate the indian market in response to growing concerns
about the regulatory environment for ayurvedic remedies.

53
 PROTOCOLS FOR TESTING THE QUALITY OF OILS, PASTE,
TABLETS, POWDER AND SYRUP
 OILS

1. Physical Properties

a) Description
b) Colour:
c) Odour
d) Viscosity
2. Test for heavy metal
a) Lead
b) Cadmium
c) Mercury
d) Arsenic

LEAD: The following method is based on the extraction of lead by solutions of dithizone.
All reagents used for the test should have as low a content of lead as practicable. All reagent
solutions should be stored in containers of borosilicate glass. Glassware should be rinsed
thoroughly with warm dilute nitric acid, followed by water.

Special Reagents

(1) Ammonia-cyanide solution Sp. – Dissolve 2 g of potassium cyanide in 15 ml of strong

ammonia solution and dilute with water to 100 ml.

(2) Ammonium citrate solution Sp. – Dissolve 40 g of citric acid in 90 ml water. Add two
drops of phenol red solution then add slowly strong ammonia solution until the solution
acquires a reddish colour. Remove any lead present by extracting the solution with 20 ml
quantities of dithizone extraction solution until the dithizone solution retains its orange-green
colour.

54
(3) Dilute standard lead solution – Dilute 10.0 ml of standard lead solution with sufficient 1
per cent v/v solution of nitric acid to produce 100.0 ml. Each ml of this solution contains 1 μg
of lead per ml.

(4) Dithizone extraction solution – Dissolve 30 mg of diphenylthiocarbazone in 1000 ml of

chloroform and add 5 ml of alcohol. Store the solution in a refrigerator. Before use, shake a
suitable volume of the solution with about half its volume of 1 per cent v/v solution of nitric
acid and discard the acid.
(5) Hydroxylamine hydrochloride solution Sp. – Dissolve 20 g of hydroxylamine
hydrochloride in sufficient water to produce about 65 ml. Transfer to separator, add five
drops of thymol blue solution, add strong ammonia solution until the solution becomes
yellow. Add 10 ml of a 4 per cent w/v solution of sodium diethyldithiocarbamate and allow
standing for five minutes. Extract with successive quantities, each of 10 ml, of chloroform
until a 5 ml portion of the extract does not assume a yellow colour when shaken with dilute
copper sulphate solution. Add dilute hydrochloric acid until the solution is pink and then
dilute with sufficient water to produce 100 ml.

(6) Potassium cyanide solution Sp. – Dissolve 50 g of potassium cyanide in sufficient water
to produce 100 ml. Remove the lead from this solution by extraction with successive
quantities, each of 20 ml of dithizone extraction solution until the dithizone solution retains
its orange-green colour. Extract any dithizone remaining in the cyanide solution by shaking
with chloroform. Dilute this cyanide solution with sufficient water to produce a solution
containing 10 g of potassium cyanide in each 100 ml.

(7) Standard dithizone solution – Dissolve 10 ml of diphenyl thiocarbazone in 1000 ml of

chloroform. Store the solution in a glass-stoppered, lead-free bottle, protected from light and
in a refrigerator.

(8) Citrate-cyanide wash solution – To 50 ml of water add 50 ml of ammonium citrate

solution Sp. and 4 ml of potassium cyanide solution Sp., mix, and adjust the pH, if necessary,
with strong ammonia solution to 9.0.

55
(9) Buffer solution pH 2.5 – To 25.0 ml of 0.2 M potassium hydrogen phthalate add 37.0 ml
of 0.1 N hydrochloric acid and dilute with sufficient water to produce 100.0 ml.

(10) Dithizone-carbon tetrachloride solution –Dissolve 10 mg of diphenylthiocarbazone in

1000 ml of carbon tetrachloride. Prepare this solution fresh for each determination.

(11) pH 2.5 wash solution – To 500 ml of a 1 per cent v/v nitric acid add strong ammonia
solution until the pH of the mixture is 2.5, then add 10 ml of buffer solution pH 2.5 and mix.

(12) Ammonia-cyanide wash solution – To 35 ml of pH 2.5 wash solution add 4 ml of

ammonia-cyanide solution Sp., and mix.

Method
Transfer the volume of the prepared sample directed in the monograph to a separator and
unless otherwise directed in monograph, adds 6 ml of ammonium citrate solution Sp., and 2
ml hydroxylamine hydrochloride solution Sp., (For the determination of lead in iron salts use
10 ml of ammonium citrate solution Sp.). Add two drops of phenol red solution and make the
solution just alkaline (red in color) by the addition of strong ammonia solution. Cool the
solution if necessary, and add 2 ml of potassium cyanide solution Sp. Immediately extract the
solution with several quantities each of 5 ml, of dithizone extraction solution, draining off
each extract into another separating funnel, until the dithizone extraction solution retains its
green colour. Shake the combinedithizone solutions for 30 seconds with 30 ml of a 1 per cent
w/v solution of nitric acid and discard the chloroform layer. Add to the solution exactly 5 ml
of standard dithizone solution and 4 ml of ammonia-cyanide solution Sp. and shake for 30
seconds; the color of the chloroform layer is of no deeper shade of violet than that of a
control made with a volume of dilute standard lead solution equivalent to the amount of lead
permitted in the sample under examination.

CADMIUM: Determination Conditions: - Reference condition: dry temperature: 100-

120ºC, maintain 20 seconds; ash temperature: 300-500ºC, maintain 20-25 seconds; atomic
temperature: 1500-1900ºC, maintain 4-5 seconds; measurement wavelength: 228.8 nm;
background calibration: deuterium lamp (D lamp) or Zeeman Effect.

56
II Preparation of Cd standard stock solution: - Measure accurately a quantity of Cd
single-element standard solution to prepare standard stock solution Cd with 2% HNO 3, which
containing 0.4 μg per ml Cd, stored at 0-5ºC. III Preparation of calibration curve: - Measure
accurately a quantity of cadmium standard stock solutions, diluted to the concentration of 1.6,
3.2, 4.8, 6.4 and 8.0 ng per ml with 2% HNO3 respectively. Pipette accurately 10 μl the
above solutions respectively, inject them into the graphite oven, determine their absorbance,
and then draw the calibration curve with absorbance as vertical axis and concentration as
horizontal ordinate.

IV Preparation of test solution: - Reference to method of ―Preparation of test solution‖ of

Pb in the above.

V Determination: - Pipette accurately 10-20 μl of the test solution and its corresponding
reagent blank solution respectively, determine their absorbance according to the above
method of ―Preparation of calibration curve‖ (If interference occurs, weigh accurately
respectively 1 ml of the standard solution, blank solution and test solution, add 1 ml of a
solution containing 1% NH4H2PO4 and 0.2% Mg (NO3)2, shake well, determine their
absorbance according to the method above, calculate the content of Cd in the test solution
from the calibration curve.

MERCURY: Determination Conditions: - Apparatus suitable hydride generator device;

reducing agent: a solution containing 0.5% sodium borohydride and 0.1% sodium hydroxide;
carrier liquid: 1% hydrochloric acid; carrier gas: nitrogen; measurement wavelength: 253.6
nm; background calibration: deuterium lamp (D lamp) or Zeeman Effect.

II Preparation of mercury standard stock solution: - Measure accurately a proper quantity

of mercury single-element standard solution to prepare standard stock solution with 2% nitric
acid solution, which containing 1.0 μg per ml Hg, stored at 0-5ºC.

III Preparation of calibration curve: - Measure accurately 0, 0.1, 0.3, 0.5, 0.7 and 0.9 ml of
mercury standard stock solution, transfer into a 50 ml volumetric flask respectively, add 40
ml 4% sulfuric acid solution and 0.5 ml of 5% potassium permanganate solution, shake well,
57
drop 5% hydroxylamine hydrochloride solution until the violet red just disappears, dilute
with 4% sulfuric acid solution to the volume, shake well. A quantity of each solution is
injected to the hydride generator device, determine the absorbance, and then plot the
calibration curve with peak area (absorbance) as vertical axis and concentration as horizontal
ordinate.

IV Preparation of test solution

Method :- Transfer 1 g of the coarse powder of the substance being examined, accurately
weighed, into a casparian flask, add 5-10 ml of the mixture solution of nitric acid solution
(HNO3) and perchloric acid (HCIO4) (4 : 1), mix well, fix a small hopper on the flask-top,
immerse overnight, heat to slake on the electric hot plate at 120-140ºC for 4-8 hours until
slaking completely, cool, add a quantity of 4% sulfuric acid solution and 0.5 ml of 5%
potassium permanganate solution, shake well, drop 5% hydroxylamine hydrochloride
solution until the violet red colour just disappears, dilute with 4% H 2SO4 solution to 25 ml,
shake well, centrifugate if necessary, the supernatant is used as the test solution. Prepare
synchronally the reagent blank solution based on the same procedure.

V Determination :- Pipette accurately a quantity of the test solution and its corresponding
reagent blank solution, respectively, proceed as described under ―Preparation of calibration
curve‖ beginning at the words ―add 1 ml of 25% potassium iodide solution‖. Calculate the
content of mercury (Hg) in the test solution from the calibration curve.

ARSENIC: Determination Conditions: - Apparatus suitable hydride generator device,

reducing agent: a solution containing 1% sodium borohydride and 0.3% sodium hydroxide;
carrier liquid: 1% hydrochloric acid; carrier gas: nitrogen; measurement wavelength: 193.7
nm; background calibration: deuterium lamp (D lamp) or Zeeman Effect.

II Preparation of As standard stock solution :- Measure accurately a quantity of As single-

element standard solution to prepare standard stock solution with 2% nitric acid solution
(HNO3), which containing 1.0 μg per ml As, stored at 0-5ºC.

58
III Preparation of calibration curve: - Measure accurately proper quantity of arsenic
standard stock solutions, diluted with 2% HNO3 to the concentration of 2, 4, 8, 12 and 16 ng
per ml respectively. Accurately transfer 10 ml of each into 25 ml volumetric flask
respectively, add 1 ml of 25% potassium iodide solution (prepared prior to use), shake well,
add 1 ml of ascorbic acid solution (prepared prior to use), shake well, dilute with
hydrochloric acid solution (20-100) to the volume, shake well, close the stopper and immerse
the flask in a water bath at 80ºC for 3 minutes. Cool, transfer proper quantities of each
solution respectively into the hydride generator device, determine the absorbance, then plot
the calibration curve with peak area (absorbance) as vertical axis and concentration as
horizontal ordinate.

IV Preparation of test solution: - Reference to method of ―Preparation of test solution‖ of

Pb in the above.

V Determination :- Pipette accurately 10 ml of the test solution and its corresponding

reagent blank solution respectively, proceed as described under ―Preparation of calibration
curve‖ beginning at the words ―add 1 ml of 25% potassium iodide solution‖. Calculate the
content of As in the test solution from the calibration curve.

3. Microbial contamination

The tests for microbial contamination are carried out on the same sample of the
preparations being examined using the above-stated media. When the quantity in a single
container is insufficient to carry out the tests, the combined contents of the two or more
containers are used to inoculate the above-stated media.

Remove the liquid from the test containers with a sterile pipette or with a sterile syringe or a
needle. Aseptically transfer the specified volume of the material from each container to a
vessel of the culture medium. Mix the liquid with the medium but do not aerate excessively.
Incubate the inoculated media for not less than 14 days, unless otherwise specified in the
monograph, at 30º to 35º in the case of fluid thioglycollate medium and at 20º to 25º in the
case of soyabean-casein digest medium. When the material being examined renders the
medium turbid so that the presence or absence of microbial growth cannot be determined
readily by visual examination, transfer suitable portions of the medium to fresh vessels of the
59
same medium between the third and seventh days after the test is started. Continue incubation
of the transfer vessels for not less than 7 additional days after the transfer and for a total of
not less than 14 days.

For oils and oily solutions : Use media to which have been added 0.1% w/v of (4-tert-
octylphenoxy) polyethoxyethanol, 1% w/v of polysorbate 80 or other suitable emulsifying
agent, in an appropriate concentration, shown not to have any antimicrobial properties under
the conditions of test. Carry out the test as described under For aqueous solutions and
suspensions.

Cultures containing oily preparations should be shaken gently each day. However, when fluid
thioglycollate medium is used for the detection of anaerobic micro-organisms, shaking or
mixing should be kept to a minimum to maintain anaerobic conditions.

For ointments : Prepare by diluting ten-fold in a sterile diluent such as fluid B or any other
aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid
mixture (Before use, test the dispersing agent to ascertain that in the concentration used it has
no significant antimicrobial effects during the time interval for all transfers). Mix 10 ml of the
fluid mixture so obtained with 80 ml of the medium and proceeds as directed under for
aqueous solutions and suspensions.

For solids: Transfer the quantity of the preparation to be examined to the quantity of medium
specified in Table 5 and mix, the conditions of incubation being the same as For aqueous
solutions and suspensions. Proceed as directed under for aqueous solutions and suspensions
beginning at the words ―When the material being examined‖. 3.8.8.5. For sterile devices : For
articles of such size and shape as permit complete immersion in not more than 1000 ml of
culture medium test the intact article, using the appropriate media, and incubating as directed
under For aqueous solutions and suspensions. For transfusion or infusion assemblies or where
the size of an item makes immersion impracticable and only the liquid pathway must be
sterile, flush the lumen of each of twenty units with a sufficient quantity of fluid
thioglycollate medium and the lumen of each of twenty units with a sufficient quantity of
soyabean-casein digest medium to yield a recovery of not less than 15 ml of each medium,
and incubate with not less than 100 ml of each of the two media as directed under For
60
aqueous solutions and suspensions. For devices in which the lumen is so small that fluid
thioglycollate medium will not pass through, substitute alternative thioglycollate medium for
the fluid thioglycollate medium and incubate that inoculated medium an aerobically. Where
the presence of the specimen being tested, in the medium interferes with the test because of
bacteriostatic or fungistatic action, rinse the article thoroughly with the minimum amount of
fluid A. Recover the rinsed fluid and test as described For sterile devices under Method A.

Observation and Interpretation of Results

At intervals during the incubation period, and at its conclusion, examine the media for
macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation
being examined passes the tests for sterility. If evidence of microbial growth is found, reserve
the containers showing this and, unless it is demonstrated by any other means that their
presence is due to causes unrelated to the preparation being examined and hence that the tests
for sterility are invalid and may therefore be recommenced, perform a retest using the same
number of samples, volumes to be tested and the media as in the original test. If no evidence
of microbial growth is then found, the preparation being examined passes the tests for
sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they
are not readily distinguishable from those growing in the containers reserved in the first test,
the preparation being examined fails the tests for sterility. If they are readily distinguishable
from those growing in the containers reserved in the first test, perform a second retest using
twice the number of samples. If no evidence of microbial growth is found in the second
retest, the preparation being examined passes the tests for sterility. If evidence of growth of
any micro-organisms is found in the second retest the preparation being examined fails the
tests for sterility.

4. TOTAL BACTERIAL COUNT /FUNGAL COUNT

OBJECTIVE
Test for Total Microbial Count is provided to determine compliance with the requirements
given in individual monograph / specifications.

PRINCIPLE: Total microbial count is the estimation of the number of viable aerobic micro-
organisms present in the pharmaceutical articles of all kinds, from raw materials to the

61
finished forms.

BUFFERS AND MEDIA

1. pH 7.2 Phosphate Buffer
Stock Solution: Dissolve 34 g of Monobasic Potassium Phosphate in about 500 mL of water
contained in a 1000 mL volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of 4 % w/v
aqueous solution of Sodium Hydroxide (about 175 mL), add water to volume, and mix.
Dispense and sterilize. Store under refrigeration. For use, dilute the Stock Solution with water
in the ration of 1 to 800, and sterilize in an autoclave at 121 0 C , 15 lb pressure for about 15
min.

2. Fluid Soyabean Casein Digest Medium

Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean 3.0 g
Meal
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Distilled Water 1000 mL
Final pH after Sterilization 7.3 ± 0.2

Dissolve the solids in the water, warming slightly to effect solution. Cool to room
temperature and adjust pH. 7.3 ± 0.2 and Filter, if necessary. Distribute the media into
suitable containers and sterilize in an autoclave at 121 OC for about 15min.

3. Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium

Pancreatic Digest of Casein 20.0 g

Soy Lecithin 5.0 g
Polysorbate 20 -40 mL
Water 960 mL

62
Dissolve the pancreatic digest of casein and soy lecithin in 960 mL of water, heating in a
water batch at 48 - 50 OC for about 30 min. to effect solution. Add 40 mL of Polysorbate 20.
Mix, and dispense as desired and sterilize in an autoclave at 121 OC for about 15min..

4. Sabouraud Glucose Agar with Antibiotics

Peptones (meat and Casein) 10.0 g

D- Glucose Monohydrate 40.0 g
Agar 15.0 g
Water 1000 mL

Water Adjust the pH to 5.4 +2. Sterilize by heating in an autoclave at 1210C for 15 min.
immediately before use, add 0.10 g of Benzylpenicillin Sodium and 0.10 g of Tetracycline
per liter of medium as sterile solutions or, alternatively, add 50 mg of Chloramphenicol per
liter of medium.

5. Peptone Water

(Buffered Sodium Chloride - Peptone Solution pH 7.0)

Potassium Dihydrogen Orthophosphate 3.56 g

Disodium Hydrogen Orthophosphate 7.23 g
Sodium Chloride 4.30 g
Peptone(meat and Casein) 1.0 g
Water 1000 mL

0.1 to 1.0 % w/v Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an

autoclave at 1210C for 15 min.

6. Potato Dextrose Agar Medium

63
Cook 300 g of peeled and diced potatoes in 500 mL of water prepared by distillation, filter
through cheesecloth, add water prepared by distillation to make 1000 mL and add the
following:

Agar 15 mg
Glucose 20 mg
pH after sterilization 5.6 ± 0.2

Dissolve by heating, and sterilize.

For use, just prior to pouring the plates, adjust the melted and cooled medium to 45 0C with
Sterile Tartaric Acid solution (1 in 10) to a pH of 3.5 ± 0.1 Do not reheat the pH 3.5 medium.
All the above media should be incubated for 24 hours at 370C before use. Any contaminated
media should be discarded.

Instead of preparing media, one can also use dehydrated media of Hi media / Difco and
rehydrate the required quantity as per instructions on the bottle label, dispense in required
quantities and sterilize.

Preliminary Testing
The methods given herein are invalid unless it is demonstrated that the test specimens to
which they are applied to not, of themselves, inhibit the multiplication, under the test
conditions, of micro-organisms that may be present. Therefore, prior to doing the tests,
inoculate diluted specimens of the substance being examined, with separate viable cultures of
Escherichia coli, B. subtilis and Staphylococcus aureus. Add 1 mL of not less than 10 -3
dilution of a 24 hour broth culture of the micro-organism to the first dilution (in buffer
solution pH 7.2, Fluid Soybean-Casein Digest medium, of the test material and following the
test procedure. If the organisms fail to grow in the relevant medium the procedure should be
modified by
a. increasing the volume of diligent, the quantity of test material remaining the same,
b. incorporating a sufficient quantity of a suitable inactivating agent in the diluents or by,
64
c. combining the afore-mentioned modifications so as to permit growth in the media.
If inhibitory substances are present in the sample 0.5 % of soy lecithin and 4 % of the
Polysorbate 20 may be added to the culture medium.. Alternatively, repeat the test as
described in the previous paragraph, using fluid casein digest - soy lecithin - polysorbate 20
medium to demonstrate neutralization of preservatives or other anti-microbial agents in the
test material.

PRE - TREATMENT OF THE PREPARATION BEING EXAMINED

Use separate 10 mL or 10 g specimens for testing.

Water - Soluble Products:

Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise
prescribed, in Peptone Water or another suitable medium shown not to have anti-microbial
activity under the conditions of the test and adjust the volume to 100 mL with the same
medium. If necessary, adjust the pH to about 7.

Non - Fatty Products Insoluble in Water:

Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in
Peptone Water or another suitable medium shown not to have anti-microbial activity under
the conditions of the test and dilute to 100 mL with the same medium. If necessary divide the
preparation being examined and homogenize the suspension mechanically.

A suitable surface - active agent such as 0.1 % w/v of Polysorbate 80 may be added to assist
the suspension of poorly wettable substances. If necessary, adjust the pH of the suspension to
about 7

Fatty Products:
Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed
with 5 g of Polysorbate 20 or Polysorbate 80. If necessary, heat to not more than 40 0 C. Mix
carefully while maintaining the temperature in a water bath or in an oven. Add 85 mL of
Peptone Water or another suitable medium shown not to have anti-microbial activity n the
conditions of the test, heated to not more than 40 OC if necessary. Maintain this temperature
65
for the shortest time necessary for formation of an emulsion and in any case for not more than
30 min. If necessary, adjust the pH of the emulsion to about 7.

PROCEDURE
As per IP
1. Dissolve or suspend 10 g of the substance being examined in sufficient buffer
solution, pH 7.2, Fluid Soybean - Casein Digest Medium, or Fluid Casein Digest-Soy
lecithin - Polysorbate 20 medium to produce 100 mL.
2. Perform the test for the absence of inhibitory (anti-microbial) properties as described
under Preliminary Testing before the determination of Total Microbial Count.
3. Add the substance being examined, to the medium not more than 1 hour after
preparing the appropriate dilution‘s for inoculation.

A. Plate Method
1. This method is applicable for substances that are sufficiently soluble or translucent.
Dilute further, if necessary, the sample liquid prepared as described above, so that 1
mL will be expected to yield between 30 to 300 colonies.
2. Pipette 1 mL of the final dilution into each of two sterile petri dishes.
Immediately add to each dish 15 to 20 mL of Soybean-Casein digest agar medium
that has previously been melted and cooled to about 450C.
3. Cover the petridishes, mix the sample with the agar by tilting or rotating the dishes,
and allow the contents to solidify to room temperature.
4. Invert the petri dishes, and incubate for 48 to 72 hours at 370C.
5. After incubation examine the plates of growth, count the number of colonies, and
express the average for the two plates in terms of number of micro-organisms per g of
the substance.
If no colonies are recovered from the dishes representing the initial 1: 10 dilution of
the substance, express the results as ―less than 10 micro-organisms per g of
substance‖.

66
B. Multiple - Tube Method
1. This method is applicable for substances that are insoluble or not translucent.
Into each of the fourteen test tubes of similar size place 9 mL of sterile Fluid
Soyabean casein digest medium.
2. Arrange 12 of the tubes in four sets of three tubes each. Put aside one set of three
tubes to serve as the controls.
3. Into each of three tubes of one set [―100‖ ] and into fourth tube (A) pipette 1 mL of
the Solution of suspension of the specimen, and mix.
4. From tube A pipette 1 mL of its contents into the one remaining tube (B) not included
in a set, and mix.
5. These two tubes contain 100 mg (or 100 µl) and 10 mg (10 µl) of the specimen
respectively.
6. Into each of the second set [―10‖] of three tubes pipette 1 mL from tube A and into
each tube of the third set [―1‖] pipette 1 mL from tube B.
7. Discard the unused contents of tubes A and B.
8. Close well, and incubate all of the tubes.
9. Following the incubation period, examine the tubes for growth; the three control tubes
remain clean and the observations in the tubes containing the specimen, when
interpreted by reference to Table I. indicate the most probable number of micro-
organisms per g or per mL of specimen.

As per BP

Determine the total viable aerobic count of the preparations being examined by the
membrane filtration method, the plate count method or the serial dilution method as
prescribed. Suitable degrees of dilution should be used so that the number of colony forming
units is within the limits suggested for the method to be used.

A. Membrane Filtration
1. Use membrane filters having a normal pore size not greater than 0.45 µm and 50 nm
in diameter the effectiveness of which in retaining bacteria has been established. For

67
 example, Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic
solutions and Cellulose acetate filters for strongly alcoholic solutions.
2. The filtration apparatus and membrane are sterilized by appropriate means and are
designed so that the solution being examined can be introduced and filtered under
aseptic conditions and so as to permit the removal of the membrane for transfer to the
culture medium.
Transfer 10 mL or a quantity to each of two membrane filters and filter immediately.
If necessary, dilute the pretreated preparation so that a colony count of 10 to 100 may
be expected.
3. Wash each membrane by filtering through it three or more successive quantities , each
of approximately 100 mL, of a suitable liquid such as buffered Sodium Chloride -
peptone pH 7.0. For fatty substances, this liquid may contain a suitable surface active
agent such as polysorbate 20 or polysorbate 80. Transfer one of the membrane filters,
intended primarily for the enumerate of bacteria, to the surface of the plate of casein
soybean digest agar and the other, intended primarily for the surface of a plate of
sabouraud glucose agar with antibiotics.
4. Incubate the plates for 5 days, unless a more reliable count is obtained in a shorter
time, at 30 - 350C in the test intended to detect bacteria and at 20 - 25 OC in the test
intended to detect fungi.
Count the number of colonies that are formed. Calculate the number of micro-
organisms per gram or per milliliter of the preparation being examined, if necessary
counting bacteria and fungi separately.

B. Plate Count

i. For Bacteria
1. Using petri dishes 9 to 10 cm in diameter, add to each dish a mixture of 1 mL of the
pretreated preparation and about 15 mL of liquefied casein soyabean digest agar at not
more than 45 OC .
2. Alternatively, spread the pretreated preparation on the surface of the solidified
medium in a petri dish of the same diameter.
3. If necessary, dilute the pre-treated preparation as described above so that a colony
count of not more than 300 may be expected.
68
 4. Prepare at least two such petri dishes using the same dilution and incubate at 30 -
35OC for unless a more reliable count is obtained in a shorter time.
5. Count the number of colonies that are formed.
6. Calculate the results using plates with the greatest number of colonies but taking 300
colonies per plate as the maximum consistent with good evaluation.

ii. For Fungi

1. Using petri dishes 9 - 10 cm in diameter, add to each dish a mixture of 1 mL of the
pre-treated preparation and about 15 mL of liquefied Sabouraud glucose agar with
antibiotics at not more than 450C.
2. Alternatively, spread the pretreated preparation on the surface of the solidified
medium in a petri dish of the same diameter. If necessary, diluted the pretreated
preparation as described above so that a colony count of not more than 100 may be
expected.
3. Prepare atleast two such plates using the same dilution and incubate at 20 - 25 OC for
5 days, unless a more reliable count is obtained in a shorter time.
4. Count the colonies that are formed. Calculate the results using plates with not more
than 100 colonies.

Serial Dilution
1. Prepare a series of 12 tubes each containing 9 - 10 mL of caseing soyabean digest
broth.
2. To each of the first three tubes add 1 mL of the preparation diluted, dissolved or
homogenized in the proportion 1 in 10, as described above.
3. To the next three tubes add 1 mL of a 2 in 100 dilution of the preparation and to the
next three tubes add 1 mL of a 1 in 100 dilution of the preparation.
4. To the last three tubes add 1 mL of the diligent.
5. Incubate the tubes at 30 - 35 OC for at least 5 days.
6. The last three tubes should show no microbial growth.
7. If the reading of the results is difficult or uncertain owing to the nature of the
preparation being examined, sub-culture on a liquid or solid medium and read the
results after a further period of incubation.

69
 8. Determine the most probable number of micro-organisms per gram or per milliliter of
the preparation being examined from Table I.

As per USP
1. For specimens that are sufficiently soluble or translucent to permit use of the plate
Method, otherwise, use Multiple-tube Method.
2. With either method, first dissolve or suspend 10 g of the specimen if it is a solid, or 10
mL , accurately measured if the specimen is a liquid, in pH 7.2 Phosphate Buffer,
Fluid Soyabean-Casein Digest Medium, or Fluid Casein Digest-soy Lecithin -
Polysorbate 20 Medium to make 100 mL.
3. For viscous specimens that cannot be pipetted at this initial 1:10 dilution, dilute the
specimen until a suspension is obtained i.e., 1:50 or 1: 100 etc., that can be pipetted.
4. Perform the test for absence of inhibitory (anti-microbial) properties as described
under Preparatory Testing before the determination of Total Aerobic Microbial Count.
5. Add the specimen to the medium not more than 1 h after preparing the appropriate
dilution‘s for inoculation.

Plate Method
1. Dilute further, if necessary, the fluid so that 1mL will be expected to yield between 30
and 300 colonies.
2. Pipette 1 mL of the final dilution onto each of two sterile petridishes.
3. Promptly add to each dish 15 - 20 mL of Soyabean Casein Digest Agar Medium when
testing for bacteria and add 15 - 20 mL Sabouraud Dextrose Agar medium or Potato
Dextrose Agar medium for molds and yeasts,. that previously has been melted and
cooled to approx. 450C.
4. Cover the petridishes, mix the sample with the agar by tilting or rotating the dishes,
and allow the contents to solidify at room temperature.
5. Invert the petridishes, and incubate for 48 - 72 hours at 37 OC for bacteria and
incubate for 5 - 7 days at 20 - 250C for molds and yeasts.
6. Following incubation, examine the plates for growth, count the number of colonies,
and express the average for the two plates in terms of the number of micro-organisms
per g or per mL of specimen.

70
 7. If no microbial colonies are recovered from the dishes representing the initial 1: 10
dilution of the specimen, express the results as ―less than 10 micro-organisms per g or
per mL of specimen.‖

CALCULATIONS

Take the average count of each dilution and multiply it by the dilution factor. Calculate the
average form the readings obtained. This gives the count per gram or per mL of the sample/
Interpretation of Results :

If a limit is prescribed, it is to be interpreted as follows :

10 2 micro-organisms, MAXIMUM LIMIT OF ACCEPTANCE: 5 x 10 2

10 3 micro-organisms, MAXIMUM LIMIT OF ACCEPTANCE: 5 x 10 3

NOTE: Use the method as per the pharmacopoeial status (grade) of the material. In case of
In- house specifications, follow the method as per the IP or as specified.
5. TEST FOR SPECIFIC PATHOGEN
a) E. coli
b) salmonella spp.
c) s.aureus
d) pseudomonas aeruginosa

For specified micro-organisms: Grow separately the test strains of Staphylococcus aureus
and Pseudomonas aeruginosa in fluid soyabean-casein digest medium and Escherichia coli
and Salmonella typhimurium at 30º to 35º for 18 to 24 hours. Dilute portions of each of the
cultures using buffered sodium chloride-peptone solution pH 7.0to make test suspensions
containing about 103 viable micro-organisms per ml. Mix equal volume of each suspension
and use 0.4 ml (approximately 102 micro-organisms of each strain) as an inoculum in the test
for E. coli, salmonellae, P. aeruginosa and S.aureus, in the presence and absence of the
preparation being examined, if necessary. A positive result for the respective strain of micro-
organism should be obtained.

71
6. Pesticide Residue, Organochlorine Pesticides, Organophosphorus Pesticides,
Pyrethroids

DEFINITION: For the purposes of the Pharmacopoeia, a pesticide is any substance or

mixture of substances intended for preventing, destroying or controlling any pest, unwanted
species of plants or animals causing harm during or otherwise interfering with the production,
processing storage, transport or marketing of vegetable drugs. The item includes substances
intended for use as growth-regulators, defoliants or desiccants and any substance applied to
crops either before or after harvest to protect the commodity from deterioration during
storage and transport.

Limits: Unless otherwise indicated in the monograph, the drug to be examined at least
complies with the limits indicated in Table -1, The limits applying to pesticides that are not
listed in the table and whose presence is suspected for any reason comply with the limits set
by European Community directives 76/895 and 90/642, including their annexes and
successive updates.

Limits for pesticides that are not listed in Table-

1 nor in EC directives are calculated using the following expression:
ADI x M
————————
MDD x 100
ADI = acceptable daily intake, as published by FAO-WHO, in milligrams per kilogram of
body mass,
M = body mass in kilograms (60 kg),
MDD = daily dose of the drug, in kilograms.

If the drug is intended for the preparation of extracts, tinctures or other pharmaceutical forms
whose preparation method modifies the content of pesticides in the finished product, the
limits are calculated using the following expression:
ADI x M x E
————————
MDD x 100
72
E = extraction factor of the method of preparation, determined experimentally.
Higher limits can also be authorised, in exceptional cases, especially when a plant requires a
particular cultivation method or has a metabolism or a structure that gives rise to a higher
than normal content of pesticides. The competent authority may grant total or partial
exemption from the test when the complete history (nature and quantity of the pesticides
used, date of each treatment during cultivation and after the harvest) of the treatment of the
batch is known and can be checked precisely.

SAMPLING
Method: For containers up to 1 kg, take one sample from the total content, thoroughly
mixed, sufficient for the tests. For containers between 1 kg and 5 kg, take three samples,
equal in volume, from the upper, middle and lower parts of the container, each being
sufficient to carry out the tests. Thoroughly mix the samples and take from the mixture an
amount sufficient to carry out the tests. For containers of more than 5 kg, take three samples,
each of at least 250 g from the upper, middle and lower parts of the container. Thoroughly
mix the samples and take from the mixture an amount sufficient to carry out the tests.

Size of sampling: If the number (n) of containers is three or fewer, take samples from each
container as indicated above under Method. If the number of containers is more than three,
take n+1 samples for containers as indicated under Method, rounding up to the nearest unit if
necessary.

The samples are to be analysed immediately to avoid possible degradation of the residues. If
this is not possible, the samples are stored in airtight containers suitable for food contact, at a
temperature below 0ºC, protected from light.

Reagents: All reagents and solvents are free from any contaminants, especially pesticides
that might interfere with the analysis. It is often necessary to use special quality solvents or, if
this is not possible, solvents that have recently been re-distilled in an apparatus made entirely
of glass. In any case, suitable blank tests must be carried out.

73
Apparatus: Clean the apparatus and especially glassware to ensure that they are free from
pesticides, for example, soak for at least 16 h in a solution of phosphate-free detergent, rinse
with large quantities of distilled water R and wash with acetone and hexane or heptane.

Qualitative and quantitative analysis of pesticide residues

The analytical procedures used are validated according to the regulations in force.
In particular, they satisfy the following criteria:
– The chosen method, especially the purification steps, are suitable for the combination
pesticide residue/substance to be analyzed and not susceptible to interference from co-
extractives; the limits of detection and quantification are measured for each pesticide-matrix
combination to be analysed.
– Between 70 per cent to 110 per cent of each pesticide is recovered.
– The repeatability of the method is not less than the values indicated
– The reproducibility of the method is not less than the values indicated.
– The concentration of test and reference solutions and the setting of the apparatus are such
that a linear response is obtained from the analytical detector.

TEST FOR PESTICIDES

Organochlorine, organophosphorus and pyrethroid insecticides: The following methods

may be used, in connection with the general method above, depending on the substance being
examined, it may be necessary to modify, sometimes extensively, the procedure described
hereafter. In any case, it may be necessary to use, in addition, another column with a different
polarity or another detection method (mass spectrometer) or a different method
(immunochemical methods) to confirm the results obtained.
This procedure is valid only for the analysis of samples of vegetable drugs containing less
than 15 per cent of water. Samples with a higher content of water may be dried, provided it
has been shown that the drying procedure does not affect significantly the pesticide content.

EXTRACTION

To 10 g of the substance being examined, coarsely powdered, add 100 ml of acetone R and
allow standing for 20 mim. Add 1 ml of a solution containing 1.8 μg/ ml of carbophenothion

74
 R in toluene R. Homogenise using a high-speed blender for 3 min. Filter and wash the filter
cake with two quantities, each of 25 ml, of acetone R.

Combine the filtrate and the washings and heat using a rotary evaporator at a temperature not
exceeding 40ºC until the solvent has almost completely evaporated. To the residue add a few
milliliters of toluene R and heat again until the acetone is completely removed. Dissolve the
residue in 8 ml of toluene R. Filter through a membrane filter (45 μm), rinse the flask and the
filter with toluene R and dilute to 10.0 ml with the same solvent (solution A).

PURIFICATION

Organochlorine, organophosphorus and pyrethroid insecticides: Examine by size-

exclusion chromatography. The chromatographic procedure may be carried out using:
 A stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with styrene-
divinylbenzene copolymer R (5 μm).
 As mobile phase toluene R at a flow rate of 1 ml/min. Performance of the column.
Inject 100 μl of a solution containing 0.5 g/l of methyl red R and 0.5 g/l of oracet blue
2R R in toluene R and proceed with the chromatography. The column is not suitable
unless the colour of the eluate changes from orange to blue at an elution volume of
about 10.3 ml. If necessary calibrate the column, using a solution containing, in
toluene R, at a suitable concentration, the insecticide to be analysed with the lowest
molecular mass (for example, dichlorvos) and that with the highest molecular mass
(for example, deltamethrin). Determine which fraction of the eluate contains both
insecticides.

Purification of the test solution: Inject a suitable volume of solution A (100 μl to 500 μl)
and proceed with the chromatography. Collect the fraction as determined above (solution B).
Organophosphorus insecticides are usually eluted between 8.8 ml and 10.9 ml.
Organochlorine and pyrethroid insecticides are usually eluted between 8.5 ml and 10.3 ml.

Organochlorine and pyrethroid insecticides: In a chromatography column, 0.10 m long

and 5 mm in internal diameter, introduce a piece of defatted cotton and 0.5 g of silica gel
treated as follows: heat silica gel for chromatography R in an oven at 150ºC for at least 4 h.

75
Allow to cool and add dropwise a quantity of water R corresponding to 1.5 per cent of the
mass of silica gel used; shake vigorously until agglomerates have disappeared and continue
shaking for 2 h using a mechanical shaker. Condition the column using 1.5 ml of hexane R.
Prepacked columns containing about 0.50 g of a suitable silica gel may also be used provided
they are previously validated.

Concentrate solution B in a current of helium for chromatography R or oxygenfree nitrogen R

almost to dryness and dilute to a suitable volume with toluene R (200 μl to 1 ml according to
the volume injected in the preparation of solution B). Transfer quantitatively onto the column
and proceed with the chromatography using 1.8 ml of toluene R as the mobile phase. Collect
the eluate (solution C).

QUANTITATIVE ANALYSIS

1. Organophosphorus insecticides: Examine by gas chromatography,using

carbophenothion R as internal standard. It may be necessary to use a second internal
standard to identify possible interference with the peak corresponding to
carbophenothion.
2. Test solution: Concentrate solution B in a current of helium for chromatography R
almost to dryness and dilute to 100 μl with toluene R.
3. Reference solution: Prepare at least three solutions in toluene R containing the
insecticides to be determined and carbophenothion at concentrations suitable for
plotting a calibration curve.
4. The chromatographic procedure may be carried out using:
 A fused-silica column 30 m long and 0.32 mm in internal diameter the internal
wall of which is covered with a layer 0.25 μm thick of poly (dimethyl)
siloxane R.
 Hydrogen for chromatography R as the carrier gas. Other gases such as helium
for chromatography R or nitrogen for chromatography R may also be used
provided the chromatography is suitably validated.
 A phosphorus-nitrogen flame-ionisation detector or a atomic emission
spectrometry detector.

76
Maintaining the temperature of the column at 80ºC for 1 min, then raising it at a rate of
30ºC/min to 150ºC, maintaining at 150ºC for 3 min, then raising the temperature at a rate of
4ºC/min to 280ºC and maintaining at this temperature for 1 min and maintaining the
temperature of the injector port at 250ºC and that of the detector at 275ºC. Inject the chosen
volume of each solution. When the chromatograms are recorded in the prescribed conditions,
the relative retention times are approximately those listed in Table-3. Calculate the content of
each insecticide from the peak areas and the concentrations of the solutions.

3.2. Organochlorine and pyrethroid insecticides

Examine by gas chromatography, using carbophenothion as the internal standard. It may be
necessary to use a second internal standard to identify possible interference with the peak
corresponding to carbophenothion.

Test solution. Concentrate solution C in a current of helium for chromatography R or

oxygen-free nitrogen R almost to dryness and dilute to 500 μl with toluene R. Reference
solution. Prepare at least three solutions in toluene R containing the insecticides to be
determined and carbophenothion at concentrations suitable for plotting a calibration curve.

The chromatographic procedure may be carried out using:

– A fused silica column 30 m long and 0.32 mm in internal diameter the internal wall of
which is covered with a layer 0.25 μm thick of poly (dimethyl) (diphenyl) siloxane R.
– Hydrogen for chromatography R as the carrier gas. Other gases such as helium for
chromatography R or nitrogen for chromatography R may also be used, provided the
chromatography is suitably validated.
– An electron-capture detector.
– A device allowing direct cold on-column injection. maintaining the temperature of the
column at 80ºC for 1 min, then raising it at a rate of 30ºC/min to 150ºC, maintaining at 150ºC
for 3 min, then raising the temperature at a rate of 4ºC/min to 280ºC and maintaining at this
temperature for 1 min and maintaining the temperature of the injector port at 250ºC and that
of the detector at 275ºC. Inject the chosen volume of each solution.

77
 PASTE

1. Physical properties
a) Description
b) Colour
c) Taste

2. Total – ash Incinerate about 2 to 3 g accurately weighed, of the ground drug in a

tared platinum or silica dish at a temperature not exceeding 450º until free from
carbon, cool and weigh. If a carbon free ash cannot be obtained in this way, exhaust
the charred mass with hot water, collect the residue on an ash less filter paper,
incinerate the residue and filter paper, add the filtrate, evaporate to dryness, and ignite
at a temperature not exceeding 450º. Calculate the percentage of ash with reference to
the air-dried drug.

3. Acid – insoluble ash: Boil the ash obtained for 5 minutes with 25 ml of dilute
hydrochloric acid; collect the insoluble matter in a Gooch crucible or on an ash less
filter paper, wash with hot water and ignite to constant weight. Calculate the
percentage of acid-insoluble ash with reference to the air dried drug.

4. Total sugar: Heat 5 g with 1 ml of dilute sulphuric acid for five minutes on a
waterbath. Add 2 ml of dilute sodium hydroxide solution and 1 ml of copper sulphate
solution. A clear, blue coloured solution is produced. Continue heating on the water-
bath for five minutes. The solution remains blue and no precipitate is formed

5. Test for Heavy Metals: Same as Oils

a) Lead
b) Cadmium
c) Mercury
d) Arsenic
6. Total Bacterial Count /Fungal Count: Same as Oils

7. Test for specific pathogen: Same as Oils

a) E. coli

78
 b) Salmonella spp.
c) S. aureus
d) Pseudomonas aeruginosa

8. Pesticide residue: Same as Oils

a) Organochlorine Pesticides
b) Organophosphorus Pesticides
c) Pyrethroids

 TABLETS

1. Physical properties
a) Description
b) Colour
c) Odour
2. Disintegration time
 Not more than 15 minutes
 Not more than 60 minutes – Guggulu tablets

3. ASSAY

The assay of the element being examined is tested by determing the decreased degree
of light intensity of radiation. Atomic absorption obeys the general rule for absorption
spectrophotometry. The assay is carried out by comparing the abosorbance of the test
preparation with that of the reference preparation.

Apparatus
An atomic absorption spectrophotometer consists of a light source, an atomic generator, a
monochromator and a detector system. Some are equipped with a background compensation
system and automatic sampling system, etc.

1. Light Source: A hollow-cathode discharge lamp is usually used. The cathode is made of
the element being examined.

79
2. Atomic Generator: There are four main types : flame atomizer, graphite furnace atomizer,
hydride-generated atomizer and cold vapour atomiser.

(1) Flame atomizer – It mainly consists of a nebulizer and a burner. Its function is to
nebulize the test solution into aerosol, which is mixed with combustion gas and the mixture is
introduced into the flame generated by the burner. So that the substance being examined is to
be dried, evaporated to form the ground state atoms of the element being examined. The
burning flame is generated by different mixtures of gases; acetylene-air is mostly used. By
modifying the proportion of combustion gas, the temperature of the flame can be controlled
and a better stability and a better sensitivity can be obtained.

(2) Furnace atomizer – It consists of electric furnace and a power supply. Its function is to
dry and incinerate the substance being examined. During the stage of high temperature
atomization, the ground state atoms of the element being examined are to be formed.
Graphite is commonly used as the heater. Protection gas is introduced into the furnace to
avoid oxidation and used to transfer the sample vapor.

(3) Hydride-generated atomizer – It consists of hydride generator and atomic absorption

cell. It is used for the determination of the elements such as arsenic, selenium, stannum and
antimony etc. Its function is to reduce the element to be examined in acidic medium to the
low-boiling and easily pyrolyzed hydride. And then the hydride is swept by a stream of
carrier gas into the atomic absorption cell which consists of quartz tube and heater etc., in
which the hydride is pyrolyzed by heating to form the ground-state atom.

(4) Cold vapor atomizer – It consists of a mercury vapor atomizer and an absorption cell. It
is suitable for the determination of mercury. Its function is to reduce the mercuric ion into
mercury vapor which is swept into the quartz absorption cell by carrier gas.

3. Monochromator – Its function is to separate the specified wavelength radiation from the
electromagnetic radiations eradiated from the light source. The optical path of the apparatus
should assure the good spectra resolution and has the ability to work well at the condition of
narrow spectral band (0.2 nm). The commonly used wavelength region is 190.0-900.0 nm.
80
4. Detector system – It consists of a detector, a signal processor and a recording system. It
should have relatively higher sensitivity and better stability and can follow the rapid change
of the signal absorption.

5. Background compensation system – System employed for the correction of atmospheric

effects on the measuring system. Four principles can be utilized for background
compensation: continuous spectrum sources (a deuterium lamp is often used in the UV
region), the Zeeman Effect, the self inversion phenomena and the non resonance spectrum. In
the analysis using atomic absorption spectrophotometry, the interference to the determination
caused by background and other reasons should be noticed. Changes of some experimental
conditions, such as the wavelength, the slit width, the atomrizing condition, etc., may affect
the sensitivity, the stability and the interference. If it is flame, the suitable wavelength, slit
width and flame temperature, the addition of complexing agents and releasing agents and the
use of Standard addition method may eliminate interference. If it is furnace, system, the
selection of suitable background compensation system and the addition of suitable matrix
modifying agents, etc may remove the interference. Background compensation method shall
be selected as specified in the individual monograph.

Procedure: Method (direct calibration method): Prepare not less than 3 reference
solutions of the element being examined of different concentrations, covering the range
recommended by the instrument manufacturer and add separately the corresponding reagents
as that for the test solution and prepare the blank solution with the corresponding reagents.
Measure the absorbances of the blank solution and each reference solution of different
concentrations separately, record the readings and prepare a calibration curve with the
average value of 3 readings of each concentration on the ordinate and the corresponding
concentration on the abscissa.

Prepare a test solution of the substance being examined as specified in the monograph; adjust
the concentration to fall within the concentration range of the reference solution. Measure the
absorbance 3 times, record the readings and calculate the average value. Interpolate the mean
value of the readings on the calibration curve to determine the concentration of the element.

81
When used in the test for impurities, prepare two test preparations of the same concentration
as specified in the monograph. To one of the test preparation add an amount of the reference
substance equivalent to the limit of the element specified in the monograph. Proceed as
directed above and measure this solution to give an appropriate reading a; then measure the
test preparation without the addition of the reference substance under the same condition and
record the reading b; b is not greater than (a-b).
3. Test for heavy metals: Same as Oils
a) Lead
b) Cadmium
c) Mercury
d) Arsenic
4. Microbial contamination: Same as in Oils

5. Total bacterial count /fungal count: Same as in Oils

6. Test for specific pathogen: Same as in Oils

a) E. coli
b) Salmonella spp.
c) S.aureus
d) Pseudomonas aeruginosa

7. PESTICIDE RESIDUE: Same as in Oils

a) Organochlorine pesticides
b) Organophosphorus pesticides
c) Pyrethroids

 POWDER

1. Physical properties:
a) Description
b) Colour
c) Odour
d) Taste

82
2. ASSAY OF ELEMENT (S): Same as in paste

3. LOSS ON DRYING AT 105 ºC:

Procedure set forth here determines the amount of volatile matter (i.e., water drying
off from the drug). For substances appearing to contain water as the only volatile
constituent, the procedure given below, is appropriately used.

Place about 10 g of drug (without preliminary drying) after accurately weighing

(accurately weighed to within 0.01 g) it in a tarred evaporating dish. For example, for
underground or un powdered drug, prepare about 10 g of the sample by cutting
shredding so that the parts are about 3 mm in thickness.

Seeds and fruits, smaller than 3 mm should be cracked. Avoid the use of high speed
mills in preparing the samples, and exercise care that no appreciable amount of
moisture is lost during preparation and that the portion taken is representative of the
Official sample. After placing the above said amount of the drug in the tarred
evaporating dish dry at 105º for 5 hours, and weigh. Continue the drying and
weighing at one hour interval until difference between two successive weightings
corresponds to not more than 0.25 per cent. Constant weight is reached when two
consecutive weightings after drying for 30 minutes and cooling for 30 minutes in a
desiccator, show not more than 0.01 g difference.

4. TOTAL-ASH: Incinerate about 2 to 3 g accurately weighed, of the ground drug in a

tared platinum or silica dish at a temperature not exceeding 450º until free from
carbon, cool and weigh. If a carbon free ash cannot be obtained in this way, exhaust
the charred mass with hot water, collect the residue on an ashless filter paper,
incinerate the residue and filter paper, add the filtrate, evaporate to dryness, and ignite
at a temperature not exceeding 450º. Calculate the percentage of ash with reference to
the air-dried drug.

5. ACID – INSOLUBLE ASH Boil the ash obtained for 5 minutes with 25 ml of dilute
hydrochloric acid; collect the insoluble matter in a Gooch crucible, or on an ashless
83
 filter paper, wash with hot water and ignite to constant weight. Calculate the
percentage of acid-insoluble ash with reference to the air dried drug.

6. Particle size mesh size: 125-150

7. Test for heavy metals: Same as Oils

a) Lead
b) Cadmium
c) Mercury
d) Arsenic

8. Microbial contamination: Same as in oils

9. Total Bacterial Count /Fungal Count: Same as in Oils

10. Test For Specific Pathogen: Same as in Oil

a) E. coli
b) salmonella spp.
c) s. aureus
d) pseudomonas aeruginosa

11. PESTICIDE RESIDUE: Same as in Oils

a) Organochlorine pesticides
b) Organophosphorus pesticides
c) Pyrethroids

 SYRUP
1. PHYSICAL PROPERTIES
a) Description
b) Colour
c) Odour

84
2. TOTAL – ASH: Same as in tablets

3. ACID – INSOLUBLE ASH: Same as in tablets

4. WATER-SOLUBLE EXTRACTIVE :The quantitative tests e.g. total ash, acid-

insoluble ash, water-soluble ash, alcohol soluble extractive, water- soluble extractive,
ether-soluble extractive, moisture content, volatile oil content and assays are the
methods upon which the standards of Pharmacopoeia depend. The methods for
assays are described in their respective monographs and for other quantitative tests,
methods are not repeated in the text of monographs but only the corresponding
reference of appropriate appendix is given. The analyst is not precluded from
employing an alternate method in any instance if he is satisfied that the method,
which he uses, will give the same result as the Pharmacopoeial Method. In suitable
instances the methods of microanalysis, if of equivalent accuracy, may be substituted
for the tests and assays described. However, in the event of doubt or dispute the
methods of analysis of the Pharmacopoeia are alone authoritative.

5. ALCOHOL – SOLUBLE EXTRACTIVE: The quantitative tests e.g. total ash,

acid-insoluble ash, water-soluble ash, alcohol soluble extractive, water- soluble
extractive, ether-soluble extractive, moisture content, volatile oil content and assays
are the methods upon which the standards of Pharmacopoeia depend. The methods
for assays are described in their respective monographs and for other quantitative
tests, methods are not repeated in the text of monographs but only the corresponding
reference of appropriate appendix is given. The analyst is not precluded from
employing an alternate method in any instance if he is satisfied that the method,
which he uses, will give the same result as the Pharmacopoeial Method. In suitable
instances the methods of microanalysis, if of equivalent accuracy, may be substituted
for the tests and assays described. However, in the event of doubt or dispute the
methods of analysis of the Pharmacopoeia are alone authoritative.

6. pH Between 3.0 and 4.0, determined in a 5.0 per cent w/v solution.

85
7. TOTAL SUGAR CONTENT Heat 5 g with 1 ml of dilute sulphuric acid for five
minutes on a waterbath. Add 2 ml of dilute sodium hydroxide solution and 1 ml of
copper sulphate solution. A clear, blue coloured solution is produced. Continue
heating on the water-bath for five minutes. The solution remains blue and no
precipitate is formed

8. VISCOSITY: Viscosity is a property of a liquid, which is closely related to the

resistance to flow. In C.G.S. system, the dynamic viscosity (n) of a liquid is the
tangential force in dryness per square centimeter exerted in either of the two parallel
planes placed, 1 cm apart when the space between them is filled with the fluid and
one of the plane is moving in its own plane with a velocity of 1 cm per second
relatively to the other. The unit of dynamic viscosity is the poise (abbreviated p). The
centi poise (abbreviated cp) is 1/100th of one poise. While on the absolute scale,
viscosity is measured in poise or centi poise, it is most convenient to use the
kinematic scale in which the units are stokes (abbreviated S) and centi-stokes
(abbreviated CS). The centistokes is 1/100th of one stoke. The kinematic viscosity of
a liquid is equal to the quotient of the dynamic viscosity and the density of the liquid
at the same temperature. Viscosity of liquid may be determined by any method that
will measure the resistance to shear offered by the liquid.

Absolute viscosity can be measured directly if accurate dimensions of the measuring

instruments are known but it is more common practice to calibrate the instrument with
a liquid of known viscosity and to determine the viscosity of the unknown fluid by
comparison with that of the known.

9. Procedure — The liquid under test is filled in a U tube viscometer in accordance

with the expected viscosity of the liquid so that the fluid level stands within 0.2 mm
of the filling mark of the viscometer when the capillary is vertical and the specified
temperature is attained by the test liquid. The liquid is sucked or blown to the
specified weight of the viscometer and the time taken for the meniscus to pass the two
specified marks is measured. The kinematic viscosity in centistokes is calculated from
the following equation:

86
 Kinematic viscosity = kt

Where k = the constant of the viscometer tube determined by observation on liquids

of known kinematic viscosity. T = time in seconds for meniscus to pass through the
two specified marks.

10. TEST FOR HEAVY METALS: Same as Oils

a) Lead
b) Cadmium
c) Mercury
d) Arsenic
11. MICROBIAL CONTAMINATION: Same as in oils

12. TOTAL BACTERIAL COUNT /FUNGAL COUNT: Same as in Oils

13. TEST FOR SPECIFIC PATHOGEN: Same as in Oils

a) E. coli
b) Salmonella spp.
c) S. aureus
d) Pseudomonas aeruginosa
14. PESTICIDE RESIDUE: Same as in Oils
a) Organochlorine Pesticides
b) Organophosphorus Pesticides
c) Pyrethroids

87
 RESULTS
 OILS

1. TEST FOR HEAVY METALS

LEAD- Not more than 10 ppm
Result : 8ppm
CADMIUM- Not more than 0.3 ppm
Result : 0.1ppm
MERCURY- Not more than 01 ppm
Result : 0.5ppm
ARSENIC- Not more than 03 ppm
Result : 01ppm
TOTAL BACTERIAL COUNT
2. Not more than 1 x 105 CFU/gm
Result : 1 x 104 CFU/gm

TOTAL FUNGAL COUNT

Not more than 1 x 103 CFU/gm
Result : 1 x 102 CFU/gm
PESTICIDE RESIDUE
3. ORGANOCHLORINE PESTICIDES
ORGANOPHOSPHORUS PESTICIDES
PYRETHROIDS

Not more than 1 ppm

Result : 0.8 ppm
TEST FOR AFLATOXINS
4. (B1,B2,G1,G2)

Not more than B1 - 0.5 ppm

Result 0.2 ppm
Not more than G1 - 0.5 ppm

88
 Result 0.4 ppm
Not more than B2 - 0.1 ppm
Result 0.73 ppm
Not more than G2 - 0.1 ppm
Result 0.63 ppm

 PASTE

TEST FOR HEAVY METALS

1. LEAD -Not more than 10 ppm

Result : 5ppm

CADMIUM- Not more than 0.3 ppm

Result : 0.1ppm

MERCURY- Not more than 01 ppm

Result : 0.93ppm

ARSENIC - Not more than 03 ppm

Result : 01 ppm

TOTAL BACTERIAL COUNT

2. Not more than 1 x 105 CFU/gm

Result : 0.78 x 104 CFU/gm

TOTAL FUNGAL COUNT

Not more than 1 x 103 CFU/gm

Result : 0.58 x 103 CFU/gm

PESTICIDE RESIDUE

3. ORGANOCHLORINE PESTICIDES

ORGANOPHOSPHORUS PESTICIDES

89
 PYRETHROIDS

Not more than 1 ppm

Result : 0.38 ppm

 POWDER

PARTICLE SIZE MESH SIZE: 125-150

1.

TEST FOR HEAVY METALS

2. LEAD- Not more than 10 ppm

Result : 03 ppm

CADMIUM- Not more than 0.3 ppm

Result : 0.89 ppm

MERCURY- Not more than 01 ppm

Result : 0.97 ppm

ARSENIC- Not more than 03 ppm

Result : 03 ppm

TOTAL BACTERIAL COUNT: Not more than 1 x 105 CFU/gm

3. Result : 0.89 x 102 CFU/gm

TOTAL FUNGAL COUNT: Not more than 1 x 103 CFU/gm

Result : 0.73 x 103 CFU/gm

PESTICIDE RESIDUE

4. ORGANOCHLORINE PESTICIDES

ORGANOPHOSPHORUS PESTICIDES

90
 PYRETHROIDS

Not more than 1 ppm

Result : 0.85 ppm

TEST FOR AFLATOXINS (B1,B2,G1,G2)

5. B1 - Not more than 0.5 ppm

Result 0.25 ppm

G1 - 0.5 Not more than ppm

Result : 0.35 ppm

B2 - Not more than 0.1 ppm

Result : 0.21 ppm

G2 - Not more than 0.1 ppm

Result : 0.19 ppm

 SYRUP

1. pH - Between 3.0 and 4.0, determined in a 5.0 per cent w/v solution.

2. TEST FOR HEAVY METALS

LEAD- Not more than 10 ppm

Result : 8ppm
CADMIUM- Not more than 0.3 ppm
Result : 0.1ppm
MERCURY- Not more than 01 ppm
Result : 0.5ppm
ARSENIC- Not more than 03 ppm
Result : 01ppm

91
 3. TOTAL BACTERIAL COUNT

TOTAL BACTERIAL COUNT

Not more than 1 x 105 CFU/gm
Result : 1 x 104 CFU/gm

TOTAL FUNGAL COUNT

Not more than 1 x 103 CFU/gm
Result : 1 x 102 CFU/gm
4. TOTAL BACTERIAL COUNT
Not more than 1 x 105 CFU/gm
Result : 1 x 104 CFU/gm

TOTAL FUNGAL COUNT

Not more than 1 x 103 CFU/gm
Result : (1 x 102 CFU/gm)

 TABLETS

1. TEST FOR HEAVY METALS

LEAD -Not more than 10 ppm

Result : 5ppm

CADMIUM- Not more than 0.3 ppm

Result : 0.1ppm

MERCURY- Not more than 01 ppm

Result : 0.93ppm

ARSENIC - Not more than 03 ppm

Result : 01 ppm

92
2. TOTAL BACTERIAL COUNT

Not more than 1 x 105 CFU/gm

Result : 0.78 x 104 CFU/gm

TOTAL FUNGAL COUNT

Not more than 1 x 103 CFU/gm

Result : 0.58 x 103 CFU/gm

3. PESTICIDE RESIDUE

ORGANOCHLORINE PESTICIDES

ORGANOPHOSPHORUS PESTICIDES

PYRETHROIDS

Not more than 1 ppm

Result : 0.38 ppm

4. TEST FOR AFLATOXINS (B1,B2,G1,G2)

B1 - Not more than 0.5 ppm

Result 0.25 ppm

G1 - 0.5 Not more than ppm

Result : 0.35 ppm

B2 - Not more than 0.1 ppm

Result : 0.21 ppm

G2 - Not more than 0.1 ppm

Result : 0.19 ppm

93
 CONCLUSIONS
Plant materials are used throughout the developed and developing world as home remedies,
in over-the-counter drug products, and as raw material for the pharmaceutical industry, and
they represent a substantial proportion of the global drug market. Therefore, it is essential to
establish internationally recognized guidelines for assessing their quality. Certain herbs have
become popular over the years, but the public, medical practitioners and the media still have
a poor understanding of ayurvedic medicine. Evidence is emerging on the dangers of herbs.
As in most situations, the truth lies hidden under the media hype, poorly understood science,
and exaggerated claims. Seeing ayurvedic medicines as either panaceas or poisons blinds us
to the reality that in most cases they are neither! Lack of experience, information, and
education about herbs make consumers, physicians, and other orthodox health care provider‘s
easy victims of market exploitation and ayurvedic myths.

There is no rational reason behind the tendency to equate ―natural‖ with ―harmlessness.‖ The
fact that something is natural does not necessarily make it safe or effective. In addition, a lack
of knowledge of phytochemistry leads to misinterpretation and misunderstanding. It is very
likely that some herbs will have side effects, interact with other medications, and be toxic.
Information on isolated constituents should not be applied directly to the whole herb and
studies on in vitro forms should not be confused with oral administration. The gold standard
for proof of efficacy for a medication is the controlled double-blind trial, which can offer
proof of activity and effectiveness. In addition to this, well-designed unblended and clinical
trials, epidemiological, animal, and phytochemical studies can provide useful information on
the ayurvedic drug. It is not uncommon for studies to be carried out on animals and the
results extrapolated to humans even though they have different metabolic processes. Many
herbs have not been subjected to this type of study. We do not fully understand how many of
these neither ayurvedic medicines work, nor do we know which component is
pharmaceutically active. Even though ayurvedic remedies may be effective, do their benefits
outweigh the risks?
With rationing looming in virtually all health care systems, the question whether ayurvedic
medicines can save money is important. Not all plant medicines are cheap. Botanicals are not
patentable (they can be patented for use); hence ayurvedic remedies are not viable candidates
for the existing drug approval processes. Pharmaceutical companies will not risk a loss, and

94
ayurvedic producers, especially in developing countries, lack the financial resources even to
consider conducting research or seeking approval. In contrast to the United States, many
European and Asian countries have taken a more holistic approach to researching the efficacy
of ayurvedic remedies.

Companies supplying standardized extracts with the greatest degree of quality control
typically offer the highest quality products. Most standardized extracts are currently made
under strict guidelines set forth by individual members of the THE DEPARTMENT OF
INDIAN SYSTEM OF MEDICINE & HOMEOPATHY as well as those proposed by The
Department of Ayush. The The Department of Ayush production of standardized extracts
serves as a model for quality control processes for all forms of ayurvedic preparations.
Ayurvedic products and nutritional supplements are not the same. Thus, some manufacturers
evade regulation of their safety.

The possibility of herb–drug interactions is important but ―under-research‖ is an issue. The

World Health Assembly in resolutions has emphasized the need to ensure the quality of
medicinal plant products by using modern control techniques and applying suitable standards.
These resolutions describe a series of tests for assessing the quality of medicinal plant
materials. The tests are designed primarily for use in national drug quality control
laboratories in developing countries, and complement those described in the international
pharmacopeia, which provide quality specifications only for the few plant materials that are
included in the Protocol for Testing of Ayurvedic, Siddha and Unani Medicines by
PHARMACOPEIAL LABORATORY FOR INDIAN MEDICINES. This manual does not
constitute a herbal pharmacopeia, but a collection of test procedures to support the
development of national standards based on local market conditions, with due regard to
existing national legislation and national and regional norms.

The test procedures cannot take account of all possible impurities. Common sense and good
pharmaceutical practice should be applied in deciding whether an unusual substance not
detectable by the prescribed tests can be tolerated. The indian pharmacopeia provides quality
specifications only for the few plant materials that are included in the WHO Model List of
Essential Drugs.

95
There is a lack of open interpretation in the area of safety and efficacy, especially for
bibliographic studies. Such interpretations are particularly relevant for ayurvedic medicinal
products because they have been used for long periods of time, sometimes over centuries, and
a wealth of literature is available. It is desirable that this documented knowledge is exploited
in order to avoid unnecessary tests with animals and clinical trials. Scientific evaluation of
the traditional knowledge is needed. In many societies much of the knowledge resides in the
hand of the healers, where oral transmission of information is the unwritten rule. In most
cases, the information is not documented. As a result, in many regions, this knowledge is
endangered because the younger generation is unwilling to carry on the profession of the
elders. Knowledge that has been refined over thousands of years of experimentation with
ayurvedic medicine is being lost. A major research opportunity in this field would be to
catalogue information on ayurvedic medicines by traditional healers in cultures where these
skills are normally transmitted through an apprentice system.

Opinion about the safety, efficacy, and the appropriateness of medicinal herbs varies widely
among medical and health professionals in countries where ayurvedic remedies are used. In
most cases the safety and efficacy of drugs of ayurvedic origin cannot be attributed to one
single chemical constituent. Various pharmaceutical particulars, including the production and
collection of the starting material and the extraction procedures, need to be assessed. Some
professionals, however, accept historical, empirical evidence as the only necessary criterion
for the efficacy of ayurvedic medicines. Others would ban all ayurvedic remedies as
dangerous or of questionable value. ayurvedic medicines have the potential for improving
public health at low cost. Phytomedicines, if combined with preventive medical practice,
could be a cost-effective, practical way to shift modern health care from treatment to
prevention.

Manufacturers and distributors should attempt to certify that the ayurvedic medicines
available to the public meet certain standards by answering questions such as: Does the
product meet recognized standards of quality? Does the label accurately reflect what is in the
product? Is the product reasonably free of contaminants such as heavy metals or pesticides?
Was the product produced and packaged under clean and safe conditions? Good
housekeeping is required to prove that a product is safe and effective. To obtain this
certification, a manufacturer must submit research-based evidence that the product does what
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it claims to do and that it does so without harming the consumer. Clinical trials should be
conducted to establish facts such as average effective dose for any drug, as well as potential
side effects a compound may cause. Recommendations on product information such as
dosage limits and any warnings should also be supplied to the consumer.

Two paradigm shifts in medicine characterize the beginning of the twenty-first century: the
gradual renunciation of the long-standing reliance on monosubstance therapy in favor of a
multidrug therapy and the transition to a new kind of multitarget therapy, through which the
interference of drugs with protective, repair, and immunostimulatory mechanisms of the
human body, rather than with single disease-causing agents, gains more and more
importance. Phytomedicine research has a good chance of contributing to these new
strategies through the development of new and better drugs for an evidence-based and
rational phytotherapy. One major concern will be to investigate the multivalent and
multitarget actions of plant constituents and standardized extracts, with the aim of
rationalizing the therapeutic superiority of many plant extracts over single isolated
constituents. Phytomedicine and chemosynthetic pharmaceutical research find themselves in
a race to develop new medicines, with fewer or no side effects, for therapeutic and preventive
application in illness for which causality-based treatments are nonexistent or imperfect.

It has now become evident that there is need for a holistic approach to health care, and the
untapped potential of traditional medicines should be utilized. However, this will not be easy,
as it requires a thorough search for medicinal plants, proper guidelines for their identification,
validation of the scientific methods of isolation of active ingredients, preclinical evaluation of
their pharmacological and toxicological profiles, and clinical evidence of their usefulness.
Clinical trials should be conducted to establish facts such as the average effective dose for
any drug, as well as potential side effects a compound may cause. In short, the ayurvedic
drugs need to be analyzed in the same way as any modern drug that is with randomized
controlled clinical trials.

As doctors and researchers continue to explore the safety and effectiveness of ayurvedic
medicines, more is learned about both their promises and their pitfalls. At the same time,
legislators at the national level should continue to press for effective laws to protect
consumers from potentially harmful ayurvedic drugs. In the mean time, your own scrutiny
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and curiosity are your best protection. Quality control for efficacy and safety of ayurvedic
products is of utmost importance. The assurance of the safety of a herbal drug requires
monitoring of the quality of the finished product as well as the quality of the consumer
information on the ayurvedic remedy.

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 BIBLIOGRAPHY
1. Manuals and handouts of SPAN Laboratories (P) Ltd.
2. The Ayurvedic Pharmacopeia of India by THE DEPARTMENT OF INDIAN
SYSTEM OF MEDICINE & HOMEOPATHY.
3. Protocol for Testing of Ayurvedic, Siddha and Unani Medicines by
PHARMACOPEIAL LABORATORY FOR INDIAN MEDICINES.
4. Production of ISM Drugs with Current Good Manufacturing Practices by THE
DEPARTMENT OF INDIAN SYSTEM OF MEDICINE & HOMEOPATHY.
5. GMP for Botanicals: Regulatory and Quality Issues on Phytomedicines by Mukherjee
P.K., Verpoorte R., Business Horizons, New Delhi, 2003.
6. Quality Control, Screening, Toxicity, and Regulation of Herbal Drugs by
Wickramasinghe M. Bandaranayake.
7. Reflections on the basic concepts of Indian pharmacology by Jan Meulenbeld, 1987
8. Indian J. Exp. Biol. 1984 – Articles by M. L. Sharma,N. Chandokhe Ray, B.J.
Ghatak, K. S. Jamwal,O.P. Gupta,G. B. Singh,M.M. Ali, R.S. Thakur,K.L. Handa,P.
R. Rao,Y. K. Sareen.
9. Sebastian Pole - Ayurvedic Medicine The Principles of Traditional Practice
10. http://www.niam.com

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