Adulteration in various tea powders

(Gandhi Institute of Technology And Management)

Project Dissertation
by

Polavarapu Bhargavi Purneshwar

Regd. No. 1216114153

Under the Guidance of

Dr. K.SUNITHA M.Pharm., Ph.D.

Thesis submitted in partial fulfilment of the

requirements for the award of the degree of

Bachelor of Pharmacy

to
GITAM Institute of Pharmacy
GITAM (Deemed to be University)

2018
 GITAM INSTITUTE OF
PHARMACY
Dr. Yellapragada Subbarao Bhavan
Rushikonda ,Visakhapatnam 530 045 (A.P)

Prof. S. Ganapaty
M. Pharm., M.Sc. (UK), Ph. D.
Principal

CERTIFICATE

The project dissertation entitled “UV SPECTROPHOTOMETRIC

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS

ESTIMATION OF CINITAPRIDE HYDROGEN TARTRATE AND

OMEPRAZOLE IN BULK AND PHARMACEUTICAL DOSAGE FORM”

submitted by Polavarapu Bhargavi Purneshwar, Regd. No. 1216114153, under the

guidance of Dr. K. Sunitha, Assistant Prof., GITAM Institute of Pharmacy,

GITAM University, being certified and approved in partial fulfilment of the

requirements for the award of the degree of Bachelor of Pharmacy.

(S. GANAPATY)
 GITAM INSTITUTE OF
PHARMACY
Dr. Yellapragada Subbarao Bhavan
Rushikonda ,Visakhapatnam 530 045 (A.P)

Assistant Prof. K Sunitha,

M.Pharm, Ph.D.

CERTIFICATE

This is to certify that the project dissertation entitled “UV

SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION

FOR THE SIMULTANEOUS ESTIMATION OF CINITAPRIDE HYDROGEN

TARTRATE AND OMEPRAZOLE IN BULK AND PHARMACEUTICAL

DOSAGE FORM” is a record of bonafide project work carried out by Polavarapu

Bhargavi Purneshwar, Regd. No. 1216114153 under my supervision during the

academic year 2017-2018.

Assistant Prof. K.
Sunitha
Research Director
 Acknowledgement

The accomplishment of this dissertation is an outstanding and overwhelming

experience and I express my deep gratitude to all those who have helped me in the

successful completion of this project.

I would like to express my sincere thanks to Prof. S. Ganapathy, Dean and

principal,

GITAM institute of pharmacy, GITAM University, Visakhapatnam for giving his

valuable

support throughout my project work.

I take the opportunity to express my deep sense of gratitude and indebtedness

to my revered guide Assistant Prof. k. sunitha, M. Pharm, Ph. D department of

Pharmacognacy and phytochemistry, GITAM institute of pharmacy, GITAM

University, Visakhapatnam for her special interest, valuable guidance, kindness and

cooperation extended to me for the successful completion of this project work. All the

guidance and advices she has given will remain as a permanent treasure. I am sincerely

indebted to her.

I also extend my gratitude to other faculty members of GITAM institute of

Pharmacy for their encouragement and concern. I would like to thank my family and

friends for their support throughout my work.

With warm regards

Polavarapu Bhargavi Purneshawar

 DECLARATION

I Polavarapu Bhargavi Purneshwar a student of B. Pharm VIII

Semester bearing Regd. No. 1216114153 of GITAM Institute of Pharmacy do

hereby declare that the project dissertation entitled “UV

SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND

VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF

CINITAPRIDE HYDROGEN TARTRATE AND OMEPRAZOLE IN

BULK AND PHARMACEUTICAL DOSAGE FORM” is a record of

bonafide project work carried out by me during the academic year 2017-2018

under the guidance of Assistant Prof. k. sunitha. This work is original and has

not been submitted in part or full to this or any other university for any degree

or diploma.

Polavarapu Bhargavi Purneshwar

 INDEX

Chapter Page No.

1. Introduction 1-4

1.1. Importance of herbal medicine

1.2. Present status of herbal medicine

2. Adulteration of drugs 4-10

2.1. Types of adulteration

2.2. Reasons of adulteration

3. Evaluation of Herbal Drugs 11-16

3.1 Organoleptic evaluation or morphological evaluation

3.2 Microscopic evaluation

3.3 Chemical evaluation

3.4 Physical evaluation

3.5 Biological evaluation

3.6. Analytical evaluation

4. Caffeine 16-21

4.1. Sources of caffeine

4.2. Typical amounts in food and beverages

4.3. Guidelines for Caffeine

4.4. Effects of higher intakes of caffeine

5. Experimentation 21-24

6. Summary and Conclusion 97

7. References 99
 INTRODUCTION

1.1. IMPORTANCE OF HERBAL MEDICINES:

Herbal medicine is still the mainstay of about 75 - 80% of the world population, mainly

in the developing countries, for primary health care (1). This is primarily because of the

general belief that herbal drugs are without any side effects besides being cheap and

locally available (2). According to the World Health Organization (WHO), the use of

herbal remedies throughout the world exceeds that of the conventional drugs by two to

three times (3). The use of plants for healing purposes predates human history and forms

the origin of much modern medicine. Many conventional drugs originated from plant

sources: a century ago, most of the few effective drugs were plant based. Examples

include aspirin (willow bark), digoxin (from foxglove), quinine (from cinchona bark),

and morphine (from the opium poppy) (4). Medical history from the beginning of time

is filled with descriptions of persons who used herbs to heal the sick of the society.

However, parallel to the onset of the industrial revolution we witnessed the rise of

allopathic medicine. Herbal medicine was also an effective healing method, but was

viewed less enthusiastically (5). Herbal products were discarded from conventional

medical use in the mid-20th century, not necessarily because they were ineffective but

because they were not as economically profitable as the newer synthetic drugs (6). In

the early 19th century, scientific methods become more advanced and preferred, and

the practice of botanical healing was dismissed as quackery. In the 1960s, with concerns

over the iatrogenic effects of conventional medicine and desire for more self-reliance,

interest in “natural health” and the use of herbal products increased. Recognition of the

rising use of herbal medicines and other non-traditional remedies led to the

establishment of the office of Alternative Medicine by the National Institute of Health

1|Page
USA, in 1992. Worldwide, herbal medicine received a boost when the WHO

encouraged developing countries to use traditional plant medicine to fulfill needs

unmet by modern systems (7).

The WHO has recently defined traditional medicine (including herbal drugs) as

comprising therapeutic practices that have been in existence, often for hundreds of

years, before the development and spread of modern medicine and are still in use today.

Traditional medicine is the synthesis of therapeutic experience of generations of

practicing physicians of indigenous system of medicine. Traditional preparations

comprise medicinal plants, minerals and organic matter etc. Herbal drugs constitute

only those traditional medicines which primarily use medicinal plant preparations for

therapy. The earliest recorded evidence of their use in Indian, Chinese, Egyptian, Greek,

Roman and Syrian texts dates back to about 5000 years. The classical Indian texts

include Rigveda, Atharvaveda, Charak Samhita and Sushruta Samhita. The herbal

medicines / traditional medicaments have therefore been derived from rich traditions of

ancient civilizations and scientific heritage (8).

1.2. PRESENT STATUS OF HERBAL MEDICINE:

The wide spread use of herbal medicine is not restricted to developing countries, as it

has been estimated that 70% of all medical doctors in France and German regularly

prescribe herbal medicine (9). The number of patients seeking herbal approaches for

therapy is also growing exponentially (10). With the US Food & Drug Administration

(FDA) relaxing guidelines for the sale of herbal supplement (11), the market is booming

with herbal products (12). As per the available records, the herbal medicine market in

1991 in the countries of the European Union was about $ 6 billion (may be over $20

billion now), with Germany account for $3 billion, France $ 1.6 billion and Italy $ 0.6

2|Page
billion. In 1996, the US herbal medicine market was about $ 4 billion, which have

doubled by now. The Indian herbal drug market is about $ one billion and the export of

herbal crude extract is about $80 million (13). In the last few decades, a curious thing

has happened to botanical medicine. Instead of being killed off by medical science and

pharmaceutical chemistry, it has made come back. Herbal medicine has benefited from

the objective analysis of the medical science, while fanciful and emotional claims for

herbal cures have been thrown out, herbal treatments and plant medicine that works

have been acknowledge. And herbal medicine has been found to have some impressive

credentials. Developed empirically by trial and error, many herbal treatments were

nevertheless remarkably effective (14). In a recent survey (15) estimated that 39% of

all 520 new approved drugs in 1983-1994 were natural products or derived from natural

products and 60-80% of antibacterial and anticancer drugs were derived from natural

products (16). The penicillin that replaced mercury in the treatment of syphilis and put

an end to so many of the deadly epidemics comes from plant mold. Belladona still

provides the chemical used in opthalmological preparations and in antiseptics used to

treat gastrointestinal disorders. Rauvolfia serpentina (The Indian snake root) which has

active ingredient, reserpine, was the basic constituent of a variety of tranquilizer first

used in the 1950’s to treat certain types of emotional and mental problems. Though

reserpine is seldom used today for this purpose, its discovery was a breakthrough in the

treatment of mental illness. It is also the principal ingredient in a number of modern

pharmaceutical preparations for treating hypertension. But reserpine can have a serious

side effect-severe depression. On the other hand, tea made of R. serpentina has been

used in India as a sedative for thousands of years (17). Examination of the history of

medicine and pharmacy reveals a definite pattern. Humankind first utilized materials

found in the environment on an empirical basis to cure various ailments. These plant,

3|Page
animal parts and even microorganisms were initially employed in unmodified form,

then as concentrated extract to improve their intensity and uniformity of action.

Subsequently, pure chemical compounds as prototypes synthetic chemical entities were

developed that possessed even greater activity (18). In fact, plant substance remains the

basis for a very large proportion of the medications used today for treating heart

diseases, hypertension, depression, pain, cancer, asthma, neurological disorders,

irritable bowel syndrome, liver diseases and other ailments (19,20,21,22,23). By 1994,

pharmacologist Norman Farnswoth had identified over 119 plant-derived substances

that are used globally as drug. Many of the prescription drugs sold in United States are

molecules derived from or modeled after naturally occurring molecules in plant.

Interest in natural product research has been rekindled by discoveries of novel

molecules from marine organisms (such as bryostatin) and potent new

chemotherapeutic agents from plants (such as Taxol). Research has been facilitated by

new rapid–through put bioassays in which robotic arms and computer controlled

cameras test exceedingly small quantities of plant samples for the presence of the

compounds active against a multiplicity of disease targets. It is possible to accomplish

in a few minutes that once took months to analyze in laboratory. Even with new

technology, it appears that one of the best sources for finding plant species to test is still

the healer’s pouch, because such plants have often been tested by generations of

indigenous people. Yet at this crescendo of enthusiasm for herbal medicine, an

increasing number of aged healers are dying with their knowledge left unrecorded. Too

often though forests disappear without any notice. Currently 12.5 percent of all plant

species are threatened with immediate extinction. Most botanists regard this estimate

by the International Union for the Conservation of Nature (IUCN) as conservative,

4|Page
because it considers only species known to science; numerous undiscovered species

pass from the world unrecorded and unmourned (24).

2. ADULTERATION OF DRUGS:

Adulteration is a practice of substituting the original crude drug partially or fully with

other substances which is either free from or inferior in therapeutic and chemical

properties or addition of low grade or spoiled drugs or entirely different drug similar to

that of original drug substituted with an intention of enhancement of profits (25,26).

Adulteration may also be defined as mixing or substituting the original drug material

with other spurious, inferior, defective, spoiled, useless other parts of same or different

plant or harmful substances or drug which do not confirm with the official standards.

A drug shall be deemed to be adulterated if it consists, in whole or in part, of any filthy,

putrid or decomposed substance. Adulteration in market samples is one of the greatest

drawbacks in promotion of herbal products. Adverse Event Reports are not due to the

intended herb, but rather due to the presence of an unintended herb [27]. Medicinal plant

dealers have discovered the scientific methods in creating adulteration of such a high

quality that without microscopic and chemical analysis, it is very difficult to trace these

adulterations [28,29]

2.1 Types of Adulterants [30]

Drugs are generally adulterated or substituted with substandard, inferior or artificial

drugs.

2.1.1 Substitution with substandard commercial varieties:

Adulterants resemble the original crude drug morphologically, chemically,

therapeutically but are sub-standard in nature and cheaper in cost.

This is the most common type of adulteration.

5|Page
 2.1.2 Substitution with Superficially Similar Inferior Drugs:

Inferior drugs may or may not have any chemical or therapeutic value. They

resemble only morphologically, so due to its resemblance they are used as

adulterants.

2.1.3 Substitution with Artificially Manufactured Substance:

The drug is adulterated with the substance which has been prepared artificially.

The artificially manufactured substance resembles the original drug. This method

is followed for the costlier drugs.

2.1.4 Substitution with Exhausted Drug: The same drug is admixed, but that drug

is devoid of medicinally active substance as it has been extracted already. Mainly

volatile oil containing drugs like clove, coriander, fennel, caraway are adulterated by

this method. As it is devoid of colour and taste due to extraction, natural colour and

taste is manipulated with additives.

2.1.5 Substitution with Synthetic Chemicals to Enhance Natural Character:

Synthetic chemicals are used to enhance natural character of the exhausted drug.

Examples: citral is added to citrus oils like lemon and orange oils.

2.1.6 Presence of Vegetative Matter of Same Plant:

Some miniature plants growing along with the medicinal plants are added due to

their colour, odour, and constituents.

2.1.7 Harmful Adulterants:

Some are harmful materials as the adulterant, are collected from market waste

materials and admixed with the drug. It is done for the liquid drugs.

6|Page
2.1.8 Adulteration of Powders:

The drugs which are in the form of powders are frequently adulterated. Examples:

dextrin is added in ipecacuanha, exhausted ginger in ginger, red sanders wood in

capsicum powder and powdered bark adulterated with brick powder.

2.2 Reason of Adulteration

Confusion in Vernacular Names: In Ayurveda, Parpatta refers to Fumaria

parviflora. In Siddha, ‘Parpadagam’ refers to Mollugo pentaphylla. Owing to

the similarity in the names in traditional systems of medicine, these two herbs

are often interchanged or adulterated or substituted. Because of the popularity

of Siddha medicine in some parts of South India, traders in these regions supply

Mollugo pentaphylla as Parpatta/Parpadagam and the North Indian suppliers

supply F. parviflora. These two can be easily identified by the presence of pale

yellow to mild brown colored, thin wiry stems and small simple leaves of

Mollugo pentaphylla and black to dark brown colored, digitate leaves with

narrow segments of F. parviflora. Casuarina equisetifolia for Tamarix indica

and Aerva lanata for Berginia ciliate are some other example for adulterations

due to confusion in names [31].

2.2.1Lack of Knowledge About Authentic Source:

Nagakesar is one of the important drugs in Ayurveda. The authentic source is

Mesua ferrea. However, market samples are adulterated with flowers of

Calophyllum inophyllum. Though the authentic plant is available in plenty

throughout the Western Ghats and parts of Himalayas, suppliers are unaware

of it. There may also be some restrictions in forest collection. Due to these

reasons, C. inophyllum (which is in the plains) is sold as Nagakesar. Authentic

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 flowers can be easily identified by the presence of two-celled ovary whereas

in case of spurious flowers they are single celled [31].

2.2.2Similarity in Morphology:

Mucuna pruriens is adulterated with other similar Papilionaceae seeds having

similarity in morphology. M. utilis (sold as white variety) and M. deeringiana

(sold as bigger variety) are popular adulterants. Apart from this M.

cochinchinensis, Canavalia virosa and C. ensiformis are also sold in Indian

markets. Authentic seeds are up to 1 cm in length with shining mosaic pattern

of black and brown color on their surface. M. deeringiana and M. utilis are

bigger (1.5-2 cm) in size. While M. deeringiana is dull black and M. utilis is

white or buff colored[31].

2.2.3 Lack of Authentic Plant:

Hypericum perforatum is cultivated and sold in European markets. In India,

availability of this species is very limited. However, the abundant Indo-Nepal

species H. patulum, sold in the name of H. perforatum. Market sample is a

whole plant with flowers and it is easy to identify them taxonomically.

Anatomically, transverse section of H. perforatum stem has compressed thin

phloem, hollow pith and absence of calcium oxalate crystals. Whereas H.

patulum hasbroader phloem, partially hollow pith and presence of calcium

oxalate crystals [31].

2.2.4 Similarity in Colour:

It is well known that with course of time, drug materials get changed to or

substituted with other plant species. ‘Ratanjot’ is a recent day example.

According to the suppliers and non-timer forest product (NTFP) contractors, in

the past, roots of Ventilago madraspatana were collected from Western Ghats,

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 as the only source of ‘Ratanjot’. However, that has not been practiced now. It

is clearly known that Arnebia euchroma vareuchroma is the present source.

Similarity is in yielding a red dye, A. euchroma substitutes V. madraspatana.

Recently V. madraspatana is not found in market. Whatever is available in the

market, in the name of Ratanjot is originated from A. euchroma[31].

2.2.5 Careless Collections:

Some of the herbal adulterations are due to the carelessness of herbal collectors

and suppliers. Parmelia perlata is used in Ayurveda, Unani and Siddha. It is

also used as grocery. Market samples showed it to be admixed with other species

(P. perforata and P. cirrhata). Sometimes, Usnea sp. is also mixed with them.

Authentic plants can be identified by their thallus nature [31].

2.2.6 Need for Substitution [31,32,33] Non-availability of the drug: Substitution

for Ashtavarga Dravyas (group of 8 crude drugs). Uncertain identity of the drug:

For the herb Lakshmana different species such as Arlia quinquefolia, Ipomea

sepiaria etc are considered Cost of the drug: Kumkuma being costly herb is

substituted by Kusumbha Geographical distribution of the drug: Rasna (Pluchea

lanceolata) is used in Northern India while in southeren parts Alpinia galanga is

considered as the source. The adverse reaction of the drug: Vasa is a well-known

Rakta-Pittahara (cures bleeding disorder) drug, but due to its abortificiant

activity its utility in pregnant women is limited, instead drugs such as Laksha,

Ashoka etc are substituted.

Table 1: Commonly used substitutes in Ayurvedic drug [34,35,36]

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 Sl. Botanical name Substitute
Common name Botanical name
No. drug

Baliospermum
1. Chitrak Plumbago zeylanica Danti
montanum

Marsdenia Lannea
2. Murva Jinghini
tenacissima coromandelica

3. Bakula Mimusops elengi Kamala Nelumbo nucifera

4. Tagar Valeriana wallichii Kustha Saussrea lappa

Syzigium
Lavanga
5. Jatipatra (Aril) Myristica fragrans aromaticum

Jatiphala(fruits) Myristica fragrans

Kustha Saussrea lappa

6. Puskar mool Inula racemosa
Eranda(root) Ricinus communis

7. Chavya Piper chaba Pippali(root) Piper longum

8. Draksha Vitis vinifera Kashmari phala Gmelina arborea

Clerodendrum Solanum
9. Bharangi Kantakari
serratum xanthocarpum

10. Dhanavayasa Fagonia cretica Duralabha Alhagi

pseudalhagi

11. Ahimsa Capparis sepiaria Manakanda Alocasia indica

12. Bakula (bark) Mimusops elengi Babul (bark Acacia arabica

13. Tulasi Ocimum sanctum Nirgundi Vitex negundo

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 14. Riddhi and Hobenaria spp. Varahikanda Dioscorea

Vriddhi bulbifera

Saccharum
15. Ikshu Nala Arundo donax
officinarum

16. Kakoli Lilium polyphyllum Asvagandha Withania

somnifera

17. Kshirakakoli Fritillaria roylei Asvagandha Withania

somnifera

Semecarpus Nadi Semecarpus

18. Bhallataka
anacardium Bhallataka travancorica

Aconitum
19. Ativisha Mustaka Cyperus rotundus
heterophyllum

20. Dadim Punica granatum Vrikshamla Garcinia indica

Cinnamomum Leonotis
21. Karpua Granthi parna
camphora nepetafolia

22. Nagapuspa Mesua ferrea Padma kesar Nelumbo nucifera

Desmostachya Saccharum
23. Kusha Kasha
bipinnata spontaneum

24. Kutherika Ocimum basilicum Gramya tulasi Ocimum sanctum

25. Amlavetas Garcinia pedunculata Chukra Garcinia indica

3. Evaluation of Herbal Drugs:

Evaluation means confirmation of its identity and determination of quality and purity

of the herbal drug. Evaluation of crude drug is necessary because of three main reasons:

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biochemical variations in the drug, deterioration due to treatment and storage,

substitution and adulteration as a result of carelessness, ignorance or fraud or variability

caused by differences in growth, geographical location, and time of harvesting. 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 or monograph in herbal

pharmacopoeia [37,38,39]. The crude drug can be evaluated or identified by five

methods:

3.1 Organoleptic evaluation or morphological evaluation

It means evaluation of drug by the organs of sense (skin, eye, tongue, nose and ear) or

macroscopic evaluation and it includes evaluation of drugs by color, odor, taste, size,

shape and special feature, like touch, texture etc. it is the technique of qualitative

evaluation based on the study of morphological and sensory profile of whole drugs. eg.

The fractured surfaces in cinchona, quillia and cascara barks and quassia wood are

important characteristics. Aromatic odour of umbelliferous fruits and sweet taste of

liquorice are the examples of this type of evaluation where odor of drug depends upon

the type and quality of odourous principles (volatile oils) present. Shape of drug may

be cylindrical (sarsapilla), subcylindrical (podophyllum), conical (aconite), fusiform

(jalap) etc, size represent length, breadth, thickness, diameter etc. color means external

color which varies from white to brownish black are important diagnostic characters.

The general appearance (external marking) of the weight of a crude drug often indicates

whether it is likely to comply with prescribed standard like furrows(alternate depression

or valleys), wrinkles (fine delicate furrows), annulations (transverse rings), fissures

(splits), nodules (rounded outgrowth), scars (spot left after fall of leaves, stems or

roots). Taste is specific type of sensation felt by epithelial layer of tongue. It may be

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acidic (sour), saline (salt like), saccharic (sweetish), bitter or tasteless (possessing no

taste).

3.2 Microscopic evaluation

It involves detailed examination of the drug and it can be used to identify the organized

drugs by their known histological characters. It is mostly used for qualitative evaluation

of organized crude drugs in entire and powder forms with help of microscope

[40,41,42].

Using microscope detecting various cellular tissues, trichomes, stomata, starch

granules, calcium oxalate crystals and aleurone grains are some of important parameters

which play important role in identification of certain crude drug. Crude drug can also

be identified microscopically by cutting the thin TS (transverse section), LS

(Longitudinal section) especially in case of wood and by staining them with proper

staining reagents e.g. starch and hemicelluloses is identified by blue color with iodine

solution, all lignified tissue give pink stain with phloroglucinol and HCl etc. mucilage

is stained pink with ruthenium red can be used to distinguish cellular structure.

Microscopic evaluation also includes study of constituents in the powdered drug by the

use of chemical reagents.

Quantitative aspects of microscopy includes study of stomatal number and index,

palisade ratio, vein-islet number, size of starch grains, length of fibers etc which play

important role in the identification of drug.

3.3 Chemical evaluation

Most of drugs have definite chemical constituents to which their biological or

pharmacological activity is attributed. Qualitative chemical test are used to identify

certain drug or to test their purity. The isolation, purification, identification of active

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constituents is based on chemical methods of evaluation. Qualitative chemical test such

as acid value, saponification value etc. Some of these are useful in evaluation of resins

(acid value, sulphated ash), balsams (acid value, saponification value and bester

values), volatile oils (acetyl and ester values) and gums (methoxy determination and

volatile acidity). Preliminary phytochemical screening is a part of chemical evaluation.

These qualitative chemical tests are useful in identification of chemical constituents and

detection of adulteration.

3.4 Physical evaluation

Physical constants are sometimes taken into consideration to evaluate certain drugs.

These include moisture content, specific gravity, optical rotation, refractive, melting

point, viscosity and solubility in different solvents. All these physical properties are

useful in identification and detection of constituents present in plant.

3.5 Biological evaluation

Some drugs have specific biological and pharmacological activity which is utilized for

their evaluation. Actually this activity is due to specific type of constituents present in

the plant extract. For evaluation the experiments were carried out on both intact and

isolated organs of living animals. With the help of bioassays (testing the drugs on living

animals), strength of drug in its preparation can also be evaluated [43,44,45]. Some

important biological evaluations are as follow:

3.5.1 Antibiotic activity

Some bacteria such as Salmonella typhi, styphylococcus aureus and E. coli are used to

determine the antiseptic value (the degree of antiseptic activity e.g. phenol co-efficient

of certain drugs). The activity of antibiotics is also determined by using Klebsiella

pneumonia, Micrococcus flavus, Sarcira lutea etc. living bacteria, yeast and molds are

14 | P a g e
used to evaluate certain vitamins. Microbiological assays by cylinder plate method and

turbidimetric method are used in evaluation.

3.5.2 Antifertility activity

Antifertility drugs include contraceptives and abortificients. Contraceptive drugs are

used to prevent pregnancy and abortificient to terminate pregnancy. Female rats are

used for antifertility activity i.e. measure the pregnancy rate (antiovulation and anti-

implantation) and male rats are used for antispermatogenic activity (inhibition of

spermatogenesis) and spermicidal activity (sperm motility) of herbal drugs.

3.5.3 Hypoglycemic activity

Rabbits, rats or mice are used to test hypoglycemic activity of plant extract. Radio-

immuno assay (RIA) or Enzyme linked immunosorbate assay (ELISA) are done for

measurement of insulin levels.

3.5.4 Neuropharmacological activity

Testing the herbal drugs with effects on central and autonomic nervous system. CNS

acting drugs like cocaine (Erythroxylum coca), morphine (Papaver somniferum),

cannabinol (Cannabis sativa) are tested using rodents. For testing the herbal drugs for

their effects on ANS guinea pig ileum for antispasmodic activity, rabbit jejunum for

adrenergic activity, rat phrenicnerve-diaphragm for muscle relaxant activity, frog rectus

for skeletal muscles activity.

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3.6 Analytical evaluation

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

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

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 herbal drugs [46,47,48].

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.

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 includes microbial contamination, aflatoxins, radioactivity, and pesticide

residues. Analytical methods such as photometric analysis (UV, IR, MS, and NMR),

thin layer chromatography (TLC), high performance liquid chromatography (HPLC),

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and gas chromatography (GC) can be employed in order to establish the constant

composition of herbal preparations.

Content or assay is the most difficult area of quality control to perform, since in most

herbal drugs the active constituents are not known. Sometimes markers can be used. In

all other cases, where no active constituent or marker can be defined for the herbal 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.

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 [49].

4. CAFFEINE:

Caffeine is a natural compound found in a number of plant species including coffee, tea

and cocoa. A typical cup of coffee contains 75-100mg caffeine, whilst levels in brewed

tea and cocoa are lower50,51. Caffeine is the principal active compound in coffee, but

other compounds are also present which can make it difficult to differentiate effects of

caffeine per se from other compounds52.

The European Food Safety Authority (EFSA) in a review on the Safety of Caffeine

concluded that a moderate caffeine consumption, of around 400mg caffeine per day

(the equivalent of up to 5 cups of coffee), can be enjoyed as part of a healthy balanced

diet and an active lifestyle. Pregnant and breastfeeding women are advised to limit their

caffeine intake to 200mg per day53.

17 | P a g e
Research suggests that moderate caffeine consumption may be associated with a range

of physiological effects, including mental and physical performance.

Caffeine is a mild central nervous stimulant, and is associated with increased

alertness54. The European Food Safety Authority (EFSA) concluded that a cause and

effect relationship has been established between a 75mg serving of caffeine and both

increased attention and alertness55. Caffeine works as an adenosine receptor antagonist:

with a similar structure to adenosine, caffeine may bind to the adenosine receptors,

acting as an imposter and blocking the actions of adenosine, leading to feelings of

alertness. This effect may cause sleep disturbance in some56, but may also help in

situations that require increased alertness, e.g. night shifts, long distance driving, and

jet lag57-63.

Caffeine may also help to improve physical performance during endurance exercise.

The European Food Safety Authority (EFSA) recognized that a cause and effect

relationship has been established for caffeine intake and increased endurance

performance and endurance capacity (in both cases for 3 mg/kg body weight 1 hour

before exercise), and reduction in perceived exertion (4 mg/kg body weight 1 hour

before exercise) 64.

Research suggests that there is no involvement of the circuit of dependence in the

physiological effects of caffeine, so caffeine does not fulfil the criteria to be described

as a ‘drug of dependence’65-67.

It is important to note that the individual responses to caffeine ingestion may differ

according to genetic variability and individuals often manage their own caffeine intake

to suit their personal lifestyle68-72.

18 | P a g e
4.1 Sources of caffeine

Caffeine is an alkaloid occurring naturally in some 60 plant species, of which cocoa

beans, kola nuts, tea leaves and coffee beans are the most well-known. Other natural

sources of caffeine include yerba maté, guarana berries, guayusa, and the yaupon holly.

Caffeine is added to many popular soft drinks, and is also a component of a number of

pharmacological preparations and over-the-counter medicines including analgesics,

diet-aids, and cold/flu remedies.

4.2 Typical amounts in food and beverages

The amount of caffeine consumed in beverages varies enormously and is dependent,

for example, on the strength of the drink, and the amount consumed with cup size

playing a key role. Coffea canephora (robusta) is known to contain more caffeine

than Coffea Arabica (arabica). However, as a basic guideline an average sized cup of

soluble coffee contains approximately 65 mg caffeine, whilst a cup of roast and ground

coffee contains around 85 mg. A 30 ml espresso cup contains around 50-60 mg caffeine.

Finally, a can of cola or a cup of tea contains 25-45 mg caffeine. Tea actually contains

more caffeine than coffee on a dry weight basis, but a smaller weight of tea is generally

used to prepare a brew. Decaffeinated coffee generally provides less than 3 mg caffeine

per cup. Cocoa and chocolate contain much smaller amounts of caffeine.

Caffeine content of drinks and foods:

Drinks/Foods Volume Caffeine (mg)Mean (range)

Filtered coffee 125 ml 85 (60-135)

Espresso 30 ml 60 (35-100)

19 | P a g e
 Soluble instant coffee 125 ml 65 (35-105)

Decaffeinated coffee 125 ml 3 (1-5)

Tea (leaves or bag) 150 ml 32 (20-45)

Iced tea 330 ml 20 (10-50)

Hot chocolate 150 ml 4 (2-7)

Caffeinated soft drinks 330 ml 39 (30-48)

Sugar-free caffeinated soft

drinks 330 ml 41 (26-57)

Energy drinks 330 ml 80 (70-120)

Chocolate bar 30 g 20 (5-36)

Milk chocolate 30 g 6 (1-15)

Dark chocolate 30 g 60 (20-120)

Data adapted from Illy et al., Harland et al., and Heckman et al 73,74,75.

4.3 GUIDELINES FOR CAFFEINE:

In 2015 the European Food Safety Authority (EFSA) published their Scientific Opinion

on the Safety of Caffeine, advising that caffeine intakes from all sources up to 400 mg

per day and single doses of 200mg do not raise safety concerns for adults in the general

population.

20 | P a g e
EFSA also advised that single doses of 100 mg of caffeine may increase sleep latency

and reduce sleep duration in some adult individuals, particularly when consumed close

to bedtime.

In 1983, the EU Scientific Committee for Food (SCF) in its ‘Report of the Scientific

Committee for Food on Caffeine’ considered the health effects of caffeine

consumption76. It concluded at that time that there was no apparent reason for concern

about carcinogenic or mutagenic effects of caffeine in humans at normal levels of

intake. The data reviewed did not reveal any teratogenic effects in humans, or any

adverse effects on human reproductive function, nor did they support any association

between caffeine consumption and adverse pregnancy outcome.

It has long been acknowledged that pregnant women should moderate their caffeine

intake. EFSA, in its 2015 Scientific Opinion on Caffeine recommends that, in pregnant

or lactating women, caffeine intake should be decreased to 200mg per day from all

sources.

In recent years concerns have been raised about caffeine consumption by children and

adolescents, particularly in relation to ‘energy drinks’, which may contain other

constituents that impact on health. The EFSA Scientific Opinion on Caffeine suggested

that caffeine intakes of 3 mg/kg body weight per day provides a basis for calculating

caffeine intakes of no concern for children and adolescents.

In UK children (5-10 years) the daily intake of caffeine was 12 mg/day at the age of 7

and 24 mg at the age of 10 years. Fifty-four percent of the caffeine absorbed came from

soft drinks, 8% from tea and 38% from chocolate foods or beverages. None came from

21 | P a g e
coffee77. A U.S. study has suggested that the majority of caffeine consumed by children

and adolescents comes from other caffeinated beverages such as ‘energy’ drinks78.

4.4 Effects of higher intakes of caffeine

As with many elements of our daily diet, over-consumption may in some people lead

to unwanted side effects. Most people consume a level of food or drink that they are

comfortable with and therefore would not experience such effects. However, those who

do not self-moderate their intakes of caffeine, may experience feelings of anxiety,

hyper-activity and sleep disturbance.

Caffeine decreases the quantity of sleep and mainly the temporal organisation of slow

and REM sleep65. These effects might be modulated by individual differences in the

expression of the gene coding for the adenosine A2A receptor79,80. Caffeine has also

been reported to increase anxiety in some individuals and this effect might also be

linked to another polymorphism of the A2A receptor gene81. However, caffeine

consumption is not significantly affected by its tendency to increase anxiety, in part

because substantial tolerance develops to this effect82. The negative effects linked to

over-consumption are usually short lived once an individual returns to their regular

pattern of consumption. It is well known that these effects are more marked in some

people than in others.

In most individuals, it seems that the effects of caffeine are utilised consciously or

unconsciously in the management of mood state, and the choice of coffee/caffeine is

influenced by the interaction between the mood state before the drink and the effects

anticipated based on the content of caffeine in the drink.83,84 which would hence lead

most individuals to moderate and self-control their caffeine intake.

22 | P a g e
 5. EXPERIMENTATION:

AIM: To check for adulteration in different Tea brands collected from market.

MATERIALS AND METHODS: The different tea brands like Taj Mahal, 3 Roses

and 4 other local brands were collected from market. The caffeine content is then

determined in all these brands.

EXTRACTION OF CAFFEINE:

Accurately, weigh 10g of tea powder of each brand and place it in a 250ml beaker. Add

50ml of freshly prepared distilled water. Boil the contents for 10 minutes using a water

bath. Allow the beaker to cool at room temperature. When cooled, filter the solution

using a whatman filter paper. Take 5ml of this extract in a separating funnel and add

15ml of Di chloro methane (DCM). Shake the separating funnel vigorously and allow

it to stand for some time. Now, separate the DCM layer carefully into a beaker. Add

again 15ml of DCM into the separating funnel and repeat the procedure. Separate this

layer into the same beaker having previously collected DCM layer. Evaporate the DCM

layer using the water bath. To this beaker add 5 ml of ethanol. Transfer this into a 10ml

volumetric flask. Make up the volume to 10ml with ethanol. Determine the absorption

spectrum of this sample using UV visible spectrophotometer. The observed spectrum

for different samples is as shown in Fig.1

23 | P a g e
 2.600

2.000 B

F
Abs.

1.000

-0.055
245.00 260.00 280.00 305.00
nm.

Figure 1: Overlay absorption spectrum of various brands in methanol

A – Tea roses C – Local brand-1 E – Local brand-3

B – Taj mahal D – Local brand-2 F – Local brand-4

Figure 2: Calibration curves of caffeine in methanol

24 | P a g e
 2.500

2.000
Abs.

1.000

0.000
-0.099
242.00 260.00 280.00 300.00
nm.

Table 1: calibration curve of absorbance spectroscopy

Conc. Absorbance

0 0

0.5 0.407

1 0.795

1.5 1.198

2 1.581

2.5 1.961

3 2.344

25 | P a g e
 3

y = 0.7804x + 0.0131
R² = 0.9999

2
Absorbance

0
0 0.8 1.6 2.4 3.2
Conc. (µg/ml)

Figure 3: Calibration curves of caffeine in methanol

Determination of Water soluble extractive value:

Add 5gm of sample to a 50ml of water at 80ºc taken in a beaker. Shake well and allow

to stand for 10 minutes. Filter the solution and transfer 5ml of the filtrate to a tared

evaporating dish, 7.5 cm in diameter. Evaporate the solvent on a water-bath, continue

drying for 30 minutes. Finally, dry in a steam oven for 2 hours and weigh the residue.

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

drug.

26 | P a g e
 Table 2: water soluble extractive value (% w/w)

S.NO. Brand Names Water soluble extractive value

(% w/w)

1 3 Roses 26

2 Taj mahal 24

3 Lb-1 22

4 Lb-2 22

5 Lb-3 20

6 Lb-4 18

(Lb) – local brands

6. Summary and Conclusion:

From the data obtained above of the proposed method, it was found that the caffeine

has a calibration curve for spectra (fig-2) in the range of 0.5-3µ/ml in methanol

respectively.

Linearity graph is plotted with respective of the observations. The branded and local

tea powders are analysed and presented in spectrum(fig-1). Water soluble extractive

value is also determined(table-2). From the results we are sure that adulteration in

local branded tea powders are considerably higher than the branded tea powders.

27 | P a g e
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