intern

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MAKERERE UNIVERSITY
COLLEGE OF NATURAL SCIENCES
DEPARTMENT OF CHEMISTRY
BACHELOR OF SCIENCE IN INDUSTRIAL CHEMISTRY
FIELD ATTACHMENT REPORT 2013/2014
CARRIED OUT AT
NATURAL CHEMOTHERAPEUTICALS RESEARCH INSTITUTE
Submitted by
KWEZI MWAKA JULIUS
12/U/494
212000546
COURSE CODE: ICH 2323
University Supervisor: Miss NANTABA FLORENCE
Field supervisor: Miss NASULA NALIKA.
A field Attachment Report Submitted to the Department of Chemistry in Partial
Fulfillment of the Requirements for the Award of the Degree of Bachelor of Science in
Industrial Chemistry of Makerere University.

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DECLARATION
I…………………………………..hereby certify and declare that the information in this report is
solely my own effort, accurate and correct to the best of my knowledge.
Signature: ……………………… Date: ……………………

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APPROVAL
NATURAL CHEMOTHERAPEUTICS RESEARCH INSTITUTE
P.O. BOX , WANDEGEYA, KAMPALA.
CERTIFICATE
Certified that the internship project report on Extraction, processing and standardization of
herbal medicines is the bonfire work of Kwezi Mwaka Julius, Reg No: 12/U/494, 3rd Year
B.SCI in Industrial Chemistry of Makerere University Kampala Uganda carried out under my
supervision during 10.6.2014 to15.08.2014.
Signature……………………
NALIKA NUSULA.
FIELD SUPERVISOR.
Signature…………………………
Mr. OMOJAL FRANCIS
INTERN COORDINATOR.

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ACKNOWLEDGEMENT
I would like to thank God for His love and strength that He has rendered me to finish my
internship successfully. I thank him for the blessings he grants me in my daily life, for the good
health, healthy mind and good ideas.
I acknowledge the guidance and support of all the people whose encouragement sailed me
through the challenging times to compile this report most especially my supervisor, Miss Nasula
Nalika, Mr. Tumusiime Henry Ralph-the head of Botany section whose guidance enabled me
to compile this report and Miss Nantaba Florence-University Supervisor
My special thanks to the management of NCRI for giving me opportunity to learn and get the
real practical work experience in addition to their untiring support, devotion, assistance,
encouragement, suggestions, continuous guidance and constructive criticism throughout this
internship.

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ABSTRACT
This report is for field attachment program undertaken at Natural Chemotherapeutics Research
Institute (NCRI). It basically talks about the activities I undertook during this program which
include; plant collection, pressing in botany, then extraction techniques, spectroscopic methods
and analysis of herbal extracts in Chemistry department and finally laboratory animal handling
and blood collection from rats in Pharmacology.
This report includes physic-chemical processes involved in this are those of extractions, the
chemical composition of phytochemicals present in different plants.
It also brings out different ways of Quality control in herbal drugs and they are improved locally
This paper will also give the incidental compounds and materials in herbal plants some of which
are good while others bring bad attributes to the final products like pesticides used during
production at the farm level and other fungal attack.
This report finalizes with the challenges and opportunities identified during this program
Objectives of the training
 To develop working skills in different process sections and acquire hands-on experience
in addition to personal skills and theoretical work.
 To avail a chance to students in managing or working in processes and laboratory
institutions
 To develop students’ good interpersonal relationship at the work-place
 To give students practical exposure in the field especially in industries and research
institutions
 To enable companies identify potential employees.

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INTRODUCTION
BACKGROUND:
Natural Chemotherapeutics Research Institute (NCRI) is a Government research and
development Centre under Ministry of Health (MoH). It was founded in 1964 with a mandate to
carry out applied research on plants, animal parts and minerals with the view of justifying
therapeutic claims from traditional medicine practioners (TMPs) in Uganda. The Centre
undertakes the development of quality natural products and services for improved health care
delivery by applying both indigenous and modern technologies.
NCRI is constituted of a multidisciplinary research team of skilled and technical scientists. This
team in collaboration with other partners and stake holders ensures that the Centre fulfils its
mandate to coordinate research and development on traditional medicine.
LOCATION:
Natural Chemotherapeutic Research Institute (NCRI) is located on plot 2B Nakasero Hill Road
Wandegeya - Kampala opposite Ministry of Public Service in the same premises with its sister
laboratory the Government Analytical Laboratory
VISION
A Centre of excellence for scientific research & development in traditional and complementary
medicine.
MISSION
Research & develop to promote quality natural products and services for improved health care
and economic development by applying indigenous and modern technologies through
collaborative efforts.
GOAL
To transform and sustain the use of traditional and complementary medicine contributing
towards its integration into health care system.
ACTIVITIES
In order to obtain the mentioned goal, NCRI undertakes the activities here under:

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1. Spear heading National policy formulation in the area of traditional medicine, in
corporation with other policy agencies.
2. Standardization of products involving promotion and evaluation of their safety, efficacy
and quality. In order to achieve this, the research team undertakes the following;
1. Identification of specimens
2. Phytochemical screening of both active and inactive ingredients.
3. Finger printing of analyzed products for consistency and reproducibility of both results
and products.
4. Evaluation of efficacy derived from both chemical and pharmacological analyses.
5. Safety studies in the laboratory animal models.
6. Accelerated stability tests that give indication of the possible shelf life of the products
among others.
1. Promotion of health and prevention of diseases in the communities involved in the
production and utilization of herbal medicines.
2. Collection and documentation of ethno botanical information and its maintenance in data
banks.
3. Capacity building including education, training and advocacy.
VALUES
In carrying out its operations, the research team is guided by the principles that govern the
manner in which work is done, and then we relate with stake holders. These principals are
enshrined in the following; values excellence, innovation, transparency and integrity,
accountability, team work and encouragement of people through hands on approach.
PARTNERSHIPS
In undertaking of its activities, NCRI partners with the following; the traditional healers, the
general public, donor organizations and research institutions such as Universities, National
Agriculture Research Organization and other government institute terrains that are involved in
policy development such as Uganda National Council for Science and Technology, National
Drug Authority, National Environment Management Authority (NEMA). In addition, she also
patterns with National Museum of Kenya and the institute of traditional medicine in Tanzania

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ACHIEVEMENTS
NCRI has contributed to the development of traditional and contemporary medicine (TCM)
through analyzing several herbal products as well as constructing community centers for
traditional medicine in Iganga, Kabale, Luwero and Lira in collaboration with International
Development Research Centre. NCRI has partnerships with Public Private Partnership of Health
(PPPH), International Development Research Centre and Networks on Medicinal Plants and
Traditional Medicine. In addition, NCRI has evaluated warbugia solutions for anti-bacterial and
anti-fungal properties, carica papaya for possible anti-hypertensive applications and as diuretic
and Artemisia from which Artavol was made which is a good treatment for malaria among
others.
NCRI has trained a number of students from universities including Makerere, Kyambogo,
Mbarara and Gulu universities in the areas of natural products and laboratory management and
carried out as base line studies of past and present research activities in Uganda in the herbarium
of medicinal plants and traditional medicine. The institute has acquired an Automated
Absorption Spectrophotometer which is used to quantify the levels of macro and micro elements
in plant materials and different products produced by natives.

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2.0 CHAPTER TWO. BOTANY DEPARTMENT
Botany, also known as Plant Science, is the study of plants and their interactions with other
organisms and the environment, this includes ecological restoration and conservation. As well as
studying species that are familiar to gardeners, orchardists and farmers some botanists study
lower plants, marine and freshwater algae, cyanobacteria and fungi. (According to
http://www.otago.ac.nz/botany/study/)
At NCRI, different plant species are used for producing herbal products like tea beverages,
medicinal products which can be in powder or syrup forms, linctus’s, soap, body lotions, jelly
and other products.
2.1 IMPORTANCES OF BOTANY AT NCRI
Botany is vital at NCRI because they underpin almost all animal life on Earth by generating a
large proportion of the oxygen and food that provide humans and other organisms with aerobic
respiration with the chemical energy they need to exist. Plants, algae and cyanobacteria are the
major groups of organisms that carry out photosynthesis, a process that uses the energy of
sunlight to convert water and carbon dioxide into sugars that can be used both as a source of
chemical energy and of organic molecules that are used in the structural components of cells. As
a by-product of photosynthesis, plants release oxygen into the atmosphere, a gas that is required
by nearly all living things to carry out cellular respiration. In addition, they are influential in the
global carbon and water cycles and plant roots bind and stabilize soils, preventing
soil erosion. Plants are crucial to the future of human society as they provide food, oxygen,
medicine, and products for people, as well as creating and preserving soil
2.2 COMMON TERMS AND TOOLS USED IN BOTANY
2.2.1 COMMON TERMS;
1. Herbarium: is a place where a collection of plant specimens is preserved. These
specimens may be whole plants or plant parts.
These will usually be in a dried form mounted on a sheet but, depending upon the
material, may also be kept in alcohol or other preservative.
Herbaria are essential for the study of plant taxonomy, identifying the flora of an area,
and also preserve a historical record of change in vegetation over time.

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2. Botanical garden: is a garden dedicated to the collection, cultivation and display of a
wide range of plants labeled with their botanical names.
Each botanical garden naturally develops its own special fields of interests depending on
its personnel, location, extent, available funds, and the terms of its charter and it may
contain specialist plant collections such as cacti and succulent plants, herb gardens, plants
from particular parts of the world.
3. Pressing: this is a process by which plants (plant parts) collected in the field are spread
flat on sheets of newsprint and dried, usually in a plant press, and between absorbent
papers(newspaper).
4. Mounting: a process by which pressed plant (part(s) of plant) placed on sheets of stiff
white paper by means of glue, labeled with all essential data, such as date, time and
locality, description of the plant, altitude, code number and special habitat conditions.
5. Phytochemicals: these are various bioactive chemical compounds found in plants, as
antioxidants, considered to be beneficial to human health.
However, the term ‘phytochemical’ is generally used to describe chemicals from plants that may
affect health but not essential nutrients.
2.2.2 SOME TOOLS USED IN BOTANY
1) Field press: this is used to press the specimen immediately after collecting it.
2) Newspaper
3) Field notebook: A pocket-sized notebook which is used in the field to collect the necessary
information about the plant of interest before being collected.
4) Tie-on tags: these are used during mounting and they contain the code given to the collected
specimen by the collector after pressing it.
5) Clippers: this is a pair of secateurs.
6) Diggers: A trowel, preferably with a steel shank.
7) Scrapers: this is a large spatula which is used for scraping up mosses and lichens.
8) Collecting bags: these are Plastic bags in a couple of sizes with rubber bands to close them.
Small brown paper bags for collecting fruits, seeds and bryophytes.
9) Felt tipped pens: these are used to number bryophyte collections.
10) Hand lens

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11) Topographic maps and global positioning system (GPS): Topographic maps are necessary for
locating your position and determining altitude.
Safety gear: A hat, long-sleeved shirt and long trousers to keep the sun off, a jumper and water-
proof raincoat to keep the cold and rain off, a first-aid kit, water, food and a trip plan outlining
your intended destination/s and expected time of return left with someone who will raise help if
necessary.
2.3 GATHERING OF PLANT MATERIAL
When gathering plants, select vigorous typical specimens and avoid insect-damaged plants.
Specimens should be representative of the population but should include the range of variation of
the plants. Roots, bulbs, and other underground parts should be carefully dug up and the soil
removed with care.
Make sure the specimen includes flowers and/or fruits. It may be a good idea to collect extra
flowers and fruit for identification purposes.
In collecting large herbs, shrubs and trees, different types of foliage, flowers and fruits should be
collected from the same plant. Collect sufficient material to fill a herbarium sheet (450 x 300
mm) and still leave enough room for the label. Plants which are too large for a single sheet may
be divided and pressed as a series of sheets.
Bark and wood samples are often desirable additions when collecting woody plants. There are
special requirements for the identification of some plants. A Eucalyptus specimen where
possible, should include mature leaves, juvenile leaves, buds, fruits and bark.

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3.0 CHAPTER THREE: CHEMISTRY DEPARTMENT
3.1 INTRODUCTION TO PHYTOCHEMISTRY
Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive
properties. They are nonessential nutrients, implying that they are not required by the human
body for sustaining life. Plants produce these chemicals to protect themselves but recent
research shows that the phytochemicals can also protect humans against diseases. There are
various known phytochemicals. Some of the well-known phytochemicals are lycopene in
tomatoes, isoflavones in soy and flavonoids in fruits. And they can also be grouped into eight
major families and these are
 Flavonoids: includes Quercetin and Epicatechin. Good food sources are: berries,
grapefruit, grapes, cereals and yellow and orange vegetables;
 Isoflavones: food sources include soy and soy protein, beans and legumes;
 Isothiocyanate: food sources include broccoli, cabbage, cauliflower and watercress;
 Monoterpene: food sources include citrus fruits, especially the peel;
 Organosulfur: food sources include onions and garlic;
 Saponins: food sources include olives, soy, quinoa, aloe, beans and other cereals;
 Capsaicin: food sources include chillis and other peppers (capsicum);
 Phytosterols (plant sterols): food sources include vegetable and soy oils;
(According to http://www.energiseforlife.com/wordpress/2006/07/12/introduction-to-
phytochemicals-phytonutrients/)
However these phytochemicals work specifically and these are some of possible actions they act
upon;
Stimulation of enzymes - Indoles, which are found in cabbages, stimulate enzymes that
make the estrogen less effective and could reduce the risk for breast cancer. Other
phytochemicals, which interfere with enzymes, are protease inhibitors (soy and beans),
terpenes (citrus fruits and cherries). Antioxidant - Most phytochemicals have antioxidant
activity and protect our cells against oxidative damage and reduce the risk of developing
certain types of cancer. Phytochemicals with antioxidant activity: allyl sulfides (onions,
leeks, garlic), carotenoids (fruits, carrots), flavonoids (fruits.)

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Interference with DNA replication - Saponins found in beans interfere with the replication
of cell DNA, thereby preventing the multiplication of cancer cells. Capsaicin, found in hot
peppers, protects DNA from carcinogens. Anti-bacterial effect - The phytochemical allicin
from garlic has anti-bacterial properties. Hormonal action - Isoflavones, found in soy,
imitate human estrogens and help to reduce menopausal symptoms and osteoporosis.
Physical action - Some phytochemicals bind physically to cell walls thereby preventing the
adhesion of pathogens to human cell walls. Proanthocyanidins are responsible for the anti-
adhesion properties of cranberry. Consumption of cranberries reduces the risk of urinary
tract infections improves dental health
3.2 EXTRACTION TECHNIQUES
Extraction, as the term is used pharmaceutically, involves the separation of medicinally active
portions of plant or animal tissues from the inactive or inert components by using selective
solvents in standard extraction procedures.
General Methods of Extraction of Medicinal Plants:
3.2.1 MACERATION
In this process, the whole or coarsely powdered crude drug is placed in a stoppered
container with the solvent and allowed to stand at room temperature for a period of at least 3
days with frequent agitation until the soluble matter has dissolved. The mixture then is strained,
the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration
or decantation after standing.
Infusion
Fresh infusions are prepared by macerating the crude drug for a short period of time with
cold or boiling water. These are dilute solutions of the readily soluble constituents of crude
drugs.
Digestion
This is a form of maceration in which gentle heat is used during the process of extraction.
It is used when moderately elevated temperature is not objectionable.
3.2.2 Decoction

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In this process, the crude drug is boiled in a specified volume of water for a defined time;
it is then cooled and strained or filtered. This procedure is suitable for extracting water-soluble,
heat-stable constituents.
Percolation
This is the procedure used most frequently to extract active ingredients in the preparation
of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is
generally used.
3.2.3 Hot Continuous Extraction (Soxhlet)
In this method, the finely ground crude drug is placed in a porous bag or “thimble” made
of strong filter paper, which is placed in chamber E of the Soxhlet apparatus. The extracting
solvent in flask A is heated, and its vapors condense in condenser D. The condensed extract drips
into the thimble containing the crude drug, and extracts it by contact. When the level of liquid in
chamber E rises to the top of siphon tube C, the liquid contents of chamber E siphon into flask A.
This process is continuous and is carried out until a drop of solvent from the siphon tube does not
leave residue when evaporated. The advantage of this method, compared to previously
described methods, is that large amounts of drug can be extracted with a much smaller quantity
of solvent.
3.3 STEPS INVOLVED IN THE EXTRACTION OF MEDICINAL PLANTS
In order to extract medicinal ingredients from plant material, the following sequential steps are
involved:
Size Reduction
The dried plant material is disintegrated by feeding it into a hammer mill or a disc
pulverized which has built-in sieves. The objective for powdering the plant material is to rupture
its organ, tissue and cell structures so that its medicinal ingredients are exposed to the extraction
solvent. Furthermore, size reduction maximizes the surface area, which in turn enhances the
mass transfer of active principle from plant material to the solvent.
Extraction
Extraction of the plant material is carried out in three ways:
- Cold aqueous percolation
- Hot aqueous extraction (decoction)

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- Solvent extraction (cold or hot)
Filtration
The extract so obtained is separated out from the marc (exhausted plant material) by allowing
it to trickle into a holding tank through the built-in false bottom of the extractor, which is
covered with a filter cloth. The marc is retained at the false bottom, and the extract is received in
the holding tank. From the holding tank, the extract is pumped into a sparkler filter to remove
fine or colloidal particles from the extract.
Concentration
The enriched extract from percolators or extractors, known as miscella, is fed into a wiped
film evaporator where it is concentrated under vacuum to produce a thick concentrated extract.
The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free
from solvent. Or the concentration could be done using Rotary Evaporation which is a technique
that employs a rotary evaporator (also called a “rotavap”) in order to remove excess solvents
from samples by applying heat to a rotating vessel at a reduced pressure. An important concept
that this technique applies is that liquids boil when the vapor pressure is equal to the external
pressure or atmospheric pressure. The machine utilizes a lower pressure than atmospheric
pressure which allows solvents to boil at lower temperatures. Furthermore, the rotation increases
the surface area and therefore evaporation proceeds more rapidly.
3.4 PHYTOCHEMICAL ANALYSIS
Analyses of phytochemicals was done on extracts obtained using different solvents and each
solvent had a different way of analyzing the phytochemicals present. Extracts used depended on
the nature of phytochemicals for the example we had extracts as ether, alcohol and water extracts
which were treated as follows
3.4.1 The ether extract
This solution contains the lip soluble chemical constituents as follows; the volatile oils, lipids,
fatty acids, sterols, carotenoids, basic alkaloids, flavonic aglycones, anthracenoside aglycones
(emodins or emodols), coumarins and chlorophyll
Experimental:

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25g of powdered vegetable product is extracted with ethyl ether in a continuous extraction
apparatus (soxhlet) or by repeatedly shaking until no more residues are left after evaporation of
the ether.
The ether extracts are combined, filtered and concentrated up to 50 ml. The lipophilic
constituents are identified as follows:
Identification of volatile oils and fatty substances
The ether extract (20 ml) is placed in a flask and evaporated to dryness. When the residue has a
pleasant odour, it should be dissolved in small amounts of alcohol by repeated elutions. One part
of the alcoholic solution is evaporated to dryness. Should the residue have a characteristically
pleasant odour, the vegetable product may contain some volatile oil. In such a case, the herbal
product (10g) is distilled with water (In a Neo-Clevenger apparatus) to extract the volatile oil and
identify it (description, colour, odour, physio-chemical properties).
The quantitative and qualitative determinations of the volatile oil constituents are carried out by
means of thin-layer chromatography or better by gas chromatography. Within the alcoholic
solution, besides the volatile oil, basic alkaloids and free aglycones may also be found while the
fatty substances form the residue. That is why the rest of the alcoholic solution may be utilized in
order to identify these constituents in the same way as for the ether extract.
The identification of the constituents of the fatty substance is carried out in the residue obtained
after an alcoholic elution followed by saponification. Potassium hydroxide (10ml) in an alcoholic
solution is added to the residue and refluxed on the boiling water bath until no more oil drops are
seen on the liquid surface for 2 hours.
The alcohol is distilled off and re-dissolved in hot distilled water (20ml) and the solution is
quantitatively transferred to separating funnel. The flask is firstly washed with hot distilled water
and transferred to the same separating funnel and after cooling washed with ether. The ether
solution are transferred to the same separating funnel over the aqueous solution (alkaline because
of the potassium hydroxide added distilled in excess) with agitation to extract the unsaponifiable
lipophilic constituents.

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The ether extract (10ml) was evaporated to dryness. The residue was dissolved in acetic
anhydride (0.5ml) and then in chloroform (0.5ml). The solution is transferred to a dry tube by
means of a pipette. Concentrated sulphuric acid (2ml) was added at the bottom of the tube
(Liebermann-burchard’s reaction). At the contact zone of the two liquids a brownish-red ring or
violet ring is formed. The supernatant layer becomes green or violet denoting the presence of
sterols and triterpenes. When the solution where the reaction was performed becomes greenish
it’s divided in two portions one of them serving as reference
Identification of carotenoids
The ether extract (10ml) was evaporated to dryness and a saturated solution of antimony
trichloride in chloroform (2drops). The pigments are firstly blue and later become red. With
concentrated sulphuric acid the carotenoids become usually deep blue or bluish-green.
Identification of higher fatty acids
The alkaline aqueous solution exhaustively extracted with ether, is acidified with concentrated
hydrochloric acid (pH=3). Under these conditions, the fatty acids are released from their alkaline
salts. The aqueous acidic solution becomes opalescent. The fatty acids are extracted by shaking
the solution in a separating funnel with ethyl ether repeatedly added in small amounts. Then the
ether solution is evaporated to dry ness. If the residue is oily, fatty acids are present.
Identification of fatty acids is done by chromatograph and by determination of specific indexes.
The rest of 30ml of the ether extract is used to identify basic alkaloids free aglycones, sterols,
triterpenes, and carotenoids that may be present in the vegetable product.
3.4.2 THE ALCOHOL EXTRACT
The ethanol or methanol extracts from the defatted vegetal products may contain important
groups or natural constituents, as for example; polyphenols (tannins), reducing compounds,
alkaloid salts, polyphenol glycosides (anthracenoside, coumarins, flavonosides), sterol
glycosides (cardiotonics, saponosides), triterpene glycosides and anthocyanosides.
Experimental:

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The rest of the dry vegetable product after extraction with ethyl ether (or another polar solvent) is
extracted by refluxing in a flask, two or three times with alcohol for 20 minutes. The filtered
solutions are combined and concentrated up to 50 ml.
The chemically active extracted constituents can be identified by means of some specific
reactions within the alcoholic or hydrolyzed extract.
Reactions carried out in the alcohol extract
Identification of tannins
The alcohol extract (0.5ml) is diluted with water (1ml) and diluted solution of ferric chloride
(light yellow, 2 drops) added. The occurrence of a blackish blue color shows the presence of
garlic tannins and a green blackish color indicates catechol tannins. In case of a mixture of
catechol and garlic tannins, they can be separated with the help of styassny’s regent.
Identification of reducing compounds
The alcohol extract (0.5 ml) is diluted with water (1ml) and Fehling’s (I and II) about (0.5 ml)
solutions and heat them. A brick-red precipitate denotes the presence of reducing compounds.
Identification of alkaloid salts
The alcohol extract (20 ml) is transferred to a capsule and evaporated on a boiling water-bath or
hot sand. Add hydrochloric acid (10 %, 5 ml) to the residue containing the alkaloids as salts of
some organic acids. The alkaloids become now salts of the mineral acid. From the aqueous
solution, the alkaloids are precipitated as bases with the help of ammonia solution (pH = 8, 10%)
and then extracted with a non-polar solvent (ether, chloroform). The ether or chloroform solution
is evaporated to dryness in a capsule. The residue is dissolved in hydrochloric acid solution
(approximately 1.5 ml, 2%). The acid solution in which the alkaloids are under a salt form is
divided in three test tubes: one is the reference and in the other two test tubes, 2 drops of Mayer’s
or Bertrand’s reagents are added. The occurrence of an opalescence or yellowish-white
precipitate with Mayer’s reagent or with Bertrand’s reagent shows the presence of alkaloids.
Identification of alkaloids, quaternary bases and oxidized amines

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The defatted vegetable product (20g) is extracted with alcohol or 80% aqueous alcohol by using
the method described under alcohol extract. When 80% alcohol is used, the aqueous alcohol
extracts obtained should be concentrated up to a syrupy consistency. Hydrochloric acid is added
(10%, 8ml), stir at heat using a glass rod. Cool add sodium chloride (0.5g) and stir again. Filter
the solution (paper), wash the filter with hydrochloric acid (10%, 2 ml). From the acidic aqueous
extract, use samples (1 ml) to perform the tests with Mayer’s or Bertrand’s reagents.
The occurrence of an abundant precipitate may denote the presence of:
2. Alkaloids
3. Quaternary salts or oxidized amines
4. Confirmation of alkaloids presence
The remaining acidic aqueous extract is transferred to a small separating funnel. Add
concentrated ammonia (pH = 8) and then shake with small amounts of ether or chloroform. Ether
or chloroform extract and alkaline aqueous solution are separated. Identification of alkaloids in
ether or chloroform extract is carried out following the above described method.
5. Identification of quaternary bases and oxidized amines
Acidity (pH = 3) the alkaline solution with hydrochloric acid (10% solution) and filter paper. Use
filtered samples (1ml) and perform the tests with Mayer’s or Bertrand’s reagent. The occurrence
of precipitates denotes the presence of quaternary bases or oxidized amines.
Reactions carried out in the hydrolyzed alcohol extract
To an alcohol extract (25ml) was added hydrochloric acid (10%, 15 ml) by refluxing and heated
for 30 minutes. During the hydrolysis the solution became opalescent due to precipitation of
aglycones obtained by division of the glycoside.
After cooling, the solution was three times extracted in a separating funnel with ethyl ether
(10ml). The ether extracts were then placed together (36ml) and dehydrated with anhydrous
sodium sulphate resulting in to ether and aqueous solutions. The ether extract will serve to
identify the anthracenoside, coumarins, flavonosides, steroid glycosides and anthocyanoside by
means of series of the following characteristics of each group.
Identification of anthracenoside

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The ether extract (4ml) was concentrated to a half the volume (2ml), then ammonia solution
(25%, 2ml) is added by shaking a cherish red color of the alkaline solution indicates the presence
of anthracenoside.
Identification of coumarins derivatives
The ether extract (5ml) was evaporated to dryness. The residue was dissolved by heating in water
(2ml). The aqueous solution is divided into two equal volumes in a test tube. To one of the two
was added ammonia solution (0.5ml, 10%) while the other serves as the reference. The
occurrence of a blue or green fluorescence under U.V light deeper for the alkaline solution
indicates presence of coumarins.
Identification of steroid glycosides (cardio tonic glycosides)
The extract (10ml) was evaporated to dryness. Thee residue was dissolved successively in acetic
anhydride (0.5ml) and chloroform (0.5ml). The solutions are transferred to a dry test tube by
means of a pipette, concentrated sulphuric acid (2ml) was added at the bottom.at the separating
levels of the two liquids, reddish-brown or violet-brown ring is formed, the superior layer being
bluish-green or violet for sterols and terpenes.
Identification of cardenolide cardiac glycoside aglycones was possible by using the Kidde’s test.
The residue obtained by evaporating the ether extract (4ml) was dissolved in ethanol (2ml);
alcoholic solution (1%) of 3-5 dinitrobenzoic acid (4drops) were added. by heating a
disappearing violet color was obtained.
The presence of saponins may be detected by means of foaming test or by the hemolysis test
using the aqueous solution of the residue belonging to the unhydrolyzed solution.
Identification of flavonosides (flavone glycosides)
The extract (5ml) was evaporated to dryness. The residue was dissolved in methane (50%, 2ml)
by heating, divide into two and to one add metallic magnesium and 5drops of concentrated HCL
.the solution became red for the flavonols.
Identification of anthocyanosides (anthocyanin pigments)
If the acidic solution is red and it turns neither to violet at a neutral PH nor to green or blue in
alkaline medium, anthocyanins are present

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3.4.3 THE AQUEOUS EXTRACT
Water can extract from the vegetable products hydro-soluble constituents such as:
Glucides (oses, polyoses, polyuronides)
Glycoside (heterosides)
Alkaloids salts
After the total extraction of the vegetable products by means of ethanol or methanol has already
been carried out, the same chemical constituents can now be identified in the aqueous extract.
Extraction for water is suitable for green products as chlorophyll cannot be extracted.
Experimental;
The vegetable product extracted with ether and afterwards with alcohol is dried and extracted in
a conical flask with warm water for 20 minutes. The filtered solution is concentrated up to a
volume of 50ml.
In this once again, a part of the reaction takes place directly with in the extract, but to identify
other active principle a pervious hydrolysis is necessary.
Reactions carried out using the aqueous extract
Identification of polyuronides (pentons, mucilage, gums)
2ml of aqueous extract were added drop wise in a test tube where 10 ml of ethanol had already
been placed. If a thick precipitate is formed, it will be separated off by filtration and washed
away with alcohol then stained with specific (hemoxylin, toluidine blue, methylene blue etc.).
The occurrence of a violet or blue precipitate denotes the presence of mucilage.
Identification of the reducing compounds
The identification is performed with Fehling’s solution (I and II) under the condition described
for the alcohol extract 1ml of aqueous extract is used.
Identification of glucides (oses and polyoses)
2ml of aqueous extract are transferred to a porcelain capsule and evaporated to dryness .add 2-3
ml of concentrated sulphuric acid and allow to stand for 3-5 minutes. By dehydration, furfural
(from pentoses) or hydroymethly furfural (from hexoses) is formed. And then 3-4 drops of an
alcoholic solution saturated with thymol (Moloch’s reagent) (R). The occurrences of a red color
denote the presence of glucides (oses, polyposis).

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Identification of starch
To approximately 1ml of aqueous extract, decanted and diluted add 1-3 drops of Lugol’s solution
(R). If starch is present, thee blue color appears. If starch is present, the blue color appears.
Identification of saponins (saponosides)
Shake 2ml of diluted solution in the test tube of 16 cm diameter for 15 minutes. The occurrence
of a foam column of at least 1cm in height persisting for a minimum of 15 minutes indicates the
presence of saponins. Then the Liebermann- Burchard’s reaction (see the alcohol extract (B))
and the hemolysis test (the gelatin- blood method) are performed.
Identification of tannins
The identification of tannins is carried out in 1ml of aqueous extract with a solution of ferric
chloride (see the alcohol extract (B)). When the extract contains both types of tannins, a
hydrochloric formaldehyde solutions (Styassny’s reagent) is boiled with reflux. Under these
conditions, the catechol tannins are condensed as a red precipitate which is filtered. The solution
thus obtained is neutralized with sodium acetate and some drops of ferric chloride or ferric alum
are added. If Gallic tannins are present, a deep blue color forms.
Identification of alkaloid salts
15 ml of the aqueous extract are made alkaline with a solution of 10 % ammonia (pH = 8) and
extracted with a non-polar solvent following the same technique as for the alcohol extract.
Reactions carried out within the hydrolysed aqueous extract
For hydrolysis and identification of anthracenoside, coumarins, flavonic, sterolic and triterpene
glycosides see the techniques described under alcohol extract (B).
3.5 INSTRUMENTAL ANALYTICAL TECHNIQUES
AN OVERVIEW OF CHROMATOGRAPHY AND SPECTROSCOPY
There are basically two mostly used instrumental analytical techniques in the laboratory and
these are;
Chromatographic Techniques

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Spectroscopic Techniques
3.5.1 CHROMATOGRAPHY
A technique exploiting the interaction of the components in a mixture of a stationary phase and a
mobile phase (solvent) in order to separate them into their constituents. Components are
separated by different levels of adsorption to the stationary phase and solubility in the mobile
phase. The types of chromatography include;
6. Paper Chromatography and Thin Layer Chromatography (TLC)
7. Column Chromatography
8. Gas Liquid Chromatography (GLC)
9. High Performance Liquid Chromatography (HPLC)
3.5.1.1 Thin Layer (and Paper) Chromatography
TLC plates are inert supports (glass, plastic, aluminium) with a thin veneer of chromatographic
media (silica). This is done by applying a concentrated drop of sample with a capillary or
dropping tube to bottom of plate (origin pencil line). Then stand the plate in a sealed vessel and
carefully add solvent (keep solvent level below sample). Allow solvent to adsorb up the plate,
drawing the sample with it.
The ratio of distance travelled by the component (from origin) compared with the distance
travelled by the solvent front (from origin) is called the Retention factor (Rf ) .
3.5.1.2 Column Chromatography
A mixture is applied to a solid support in a chromatography column, and eluted by a solvent.
3.5.1.3 Gas Liquid Chromatography
A mixture is injected into a very thin “steel-jacketed” chromatography column.
3.5.1.4 High Performance Liquid Chromatography
A mixture is injected into a “steel-jacketed” chromatography column and eluted with solvent at
high pressure (4000psi or approximately 130atm).
STATIONARY PHASES
The surface of the stationary phase can be altered to create a surface with different bonding
properties in TLC, column chromatography, GLC and HPLC. These include;

25 | P a g e
 Normal polarity
 Reverse polarity
 Ion exchange
 Size exclusion
Normal polarity
Silica or alumina possess polar sites that interact with polar molecules.
Reverse polarity
If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly
with non-polar molecules.
Ion exchange
For the cation exchange silica is substituted with anionic residues that interact strongly with
cationic species (positively charged) while for anion exchange silica is substituted with cationic
residues that interact strongly with anionic species (negatively charged).
Size exclusion
Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set
size that restrict entry to molecules of a minimum size.
3.5.2 SPECTROSCOPY
It utilises the absorption and emission of electromagnetic radiation by atoms. Absorption is low
energy electrons absorb energy to move to higher energy level while emission are excited
electrons returning to lower energy states.
The Spectroscopic techniques are based on the fact that light absorbed (absorption) is directly
proportional to the concentration of the absorbing component.
3.5.2.1 Colorimetry
Colorimetry is a quantitative technique which makes use of the intensity in colour of a solution is
directly related to the concentration of the coloured species in it. Colorimetry can be used if the
substance to be analysed is coloured, or if it can be made coloured by a chemical reaction. The
concentration of the unknown solution can be estimated by the naked eye by comparing its

26 | P a g e
colour to those of a series of standard solutions prepared by successive dilution. However at low
concentrations, colour may not be detected.
3.5.2.2 Atomic absorption spectroscopy (AAS)
It measures small concentrations of metal ions in solution namely; Al, As, Au, B, Ca, Cd, Co, Cr,
Cs, Cu, Fe, Ge, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Si, Sr, Ti, V, W and Zn. It is also used by
industries in analysis of ores for metal content, quality control of metals in steel, testing water for
metals ions and analysing food and pharmaceuticals for metal ions.
Its advantage is that it is very sensitive (can detect concentrations as small as a few parts to g /
Litre) and generally very specific (set wavelength is strongly absorbed by the particular metal ion
being analysed)
The likely source of error is that another species may be absorbed at the same wavelength.
3.5.2.3 Principle of an Atomic absorption spectroscopy
The technique makes use of absorption spectrometry to assess the concentration of an analyte in
a sample. It requires standards with known analyte content to establish the relation between the
measured absorbance and the analyte concentration and relies therefore on the Beer-Lambert
Law.
In brief, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited
state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy
(radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a
particular electron transition in a particular element. In general, each wavelength corresponds to
only one element, and the width of an absorption line is only of the order of a few Pico meters
(pm), which gives the technique its elemental selectivity. The radiation flux without a sample
and with a sample in the atomizer is measured using a detector, and the ratio between the two
values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert
Law.

27 | P a g e
4.0 CHAPTER FOUR: PHARMACOLOGY
Pharmacology is the study of how drugs exert their effects on living systems. Or it can be
defined as the interaction of drug and any biological system of an organism (Mr. Henry
Tumusiime). Pharmacologists work to identify drug targets in order to learn how drugs work.
Pharmacologists also study the ways in which drugs are modified within organisms. In most of
the pharmacologic specialties, drugs are also used today as tools to gain insight into both normal
and abnormal function
Pharmacology is divided into majorly three divisions and these include;
• Pharmacokinetics
• Pharmacodynamics
• Pharmacogenomics
4.1 PHARMACOKINETICS
This refers to the reaction of what the body does to the drug.
The magnitude of the pharmacological effect of a drug depends on its concentration at the site of
action.
• Absorption
• Distribution
• Metabolism
• Excretion
• Serum drug levels
• Serum half-life
4.2 PHARMACODYNAMICS
Pharmacodynamics is the study of the biochemical and physiological effects of drugs on the
body or on microorganisms or parasites within or on the body and the mechanisms of drug action
and the relationship between drug concentration and effect. Wikipedia
It was however defined by Tumusiime as the study of the effects of drug does no the body of the
organism. Interaction of drugs with cellular proteins, such as receptors or enzymes, to control
changes in physiological function of particular organs.

28 | P a g e
• Drug-Receptor Interactions
– Binding
• Dose-Response
– Effect
• Signal Transduction
– Mechanism of action, Pathways
PHARMACOGENETICS
This refers to the area of pharmacology concerned with unusual responses to drugs caused by
genetic differences between individuals.
Responses that are not found in the general population, such as general toxic effects, allergies, or
side effects, but due to an inherited trait that produces a diminished or enhanced response to a
drug.
4.3 REASONS FOR STUDYING PHARMACOLOGY
To learn the processes involved in drug discovery and development
To learn and acquaint oneself with methods involving use of laboratory animals for research
To get to learn and master the ethics, care and animal housing & animal handling
To get to know how to extract blood from animals
For research and development of new medical devices
4.4.0 BASICS OF EXPERIMENTAL PHARMACOLOGY
4.4.1 BASICS AND PRINCIPLES OF RESEARCH
Research is a process of systematic inquiry into phenomenon categories. In this department we
looked at research on clinical pharmacology which is the application of pharmacodynamics and
pharmacokinetics to patients with diseases and now has a significant pharmacogenetics
component. Clinical pharmacologists study how drugs work, how they interact with the genome
and with other drugs, how their effects can alter the disease process, and how disease can
Alter their effects. Clinical trial design, the prevention of medication errors, and the optimization
of rational prescribing have become critical components of the work of clinical pharmacologists.

29 | P a g e
4.4.2 STANDARDISATION OF HERBAL MEDICINE
Standardization is important because of the following associated with herbal products (Kunle et
al)
1. Herbal drugs are usually mixtures of many constituents.
2. The active principle(s) is (are), in most cases unknown.
3. Selective analytical methods or reference compounds may not be available commercially.
4. Plant materials are chemically and naturally variable.
5. Chemo-varieties and chemo cultivars exist.
6. The source and quality of the raw material are variable
According to WHO (1996a and b, 1992), standardization and quality control of herbals is the
process involved in the physicochemical evaluation of crude drug covering aspects, such as
selection and handling of crude material, safety, efficacy and stability assessment of finished
product, documentation of safety and risk based on experience, provision of product information
to consumer and product promotion. Attention is normally paid to such quality indices such as:
1. Macro and microscopic examination: For Identification of right variety and search of
adulterants.
2. Foreign organic matter: This involves removal of matter other than source plant to get the
drug in pure form.
3. Ash values: These are criteria to judge the identity and purity of crude drug – Total ash,
sulphated ash, water soluble ash and acid insoluble ash etc.
4. Moisture content: Checking moisture content helps reduce errors in the estimation of the
actual weight of drug material. Low moisture suggests better stability against degradation of
product.
5. Extractive values: These are indicative weights of the extractable chemical constituents of
crude drug under different solvents environment.
6. Crude fibre: This helps to determine the woody material component, and it is a criterion for
judging purity.
7. Qualitative chemical evaluation: This covers identification and characterization of crude
drug with respect to phytochemical constituent. It employs different analytical technique to
detect and isolate the active constituents. Phytochemical screening techniques involve botanical

30 | P a g e
identification, extraction with suitable solvents, purification, and characterization of the active
constituents of pharmaceutical importance.
8. Chromatographic examination: Include identification of crude drug based on the use of
major chemical constituents as markers.
9. Quantitative chemical evaluation: To estimate the amount of the major classes of
constituents.
10. Toxicological studies: This helps to determine the pesticide residues, potentially toxic
elements, safety studies in animals like LD50 and Microbial assay to establish the absence or
presence of potentially harmful microorganisms.
4.4.2.1 PHYSICAL STANDARDIZATIONOF HERBAL DRUGS
Viscosity
Viscosity of a liquid is constant at a given temperature and is an index of its composition. Hence,
it can be used as a means of standardizing liquid drugs.
Melting point
In case of pure photochemical, melting points are very sharp and constant.
The crude drugs from plant or animal origin, containing the mixed chemicals, are described with
certain range of melting point.
Solubility
The presence of adulterant could be indicated by solubility studies
Moisture content and volatile matter
The moisture content of the drug should be minimized in order to prevent decomposition of
crude drug either due to chemical change or microbial contamination. The moisture content is
determined by heating a drug at 105˚c in an oven to a constant weight.
For the drugs containing volatile constituents, toluene distillation method is used. An example of
Aloe should have moisture content not more than 10%.
Bitterness value

31 | P a g e
Medicinal plants having strong bitter taste are therapeutically used as appetizing agents. The
bitterness is determined by comparing the threshold bitter concentration of an extract material
with that of quinine hydrochloride. The bitterness value is expressed as units equivalent to the
bitterness of a solution containing 1gm of quinine hydrochloride in 2000ml. 0.1gm of quinine
hydrochloride is dissolved in 100ml drinking water and the stock solution is prepared. Then it is
diluted and tested and compared with drug.
Bitterness value in unit per g =
2000 𝑥 𝑐
𝐴 𝑥 𝐵
Where, A = concentration of stock solution
B = volume of test solution in tube with threshold bitter concentration
C = quantity of quinine hydrochloride in the tube with threshold bitter concentration

32 | P a g e
Ash value
The residue remaining after incineration is the ash content of drug. Total ash method is used to
measure the total amount of material remaining after incineration. Acid insoluble ash is the
residue obtained after boiling the total ash with dil. HCl and igniting the remaining insoluble
matter. Water soluble ash is the difference in weight between total ash and residue after
treatment of total ash with water.
And the following can also be tested for efficiency of herbal drugs; Specific gravity, Density,
Optical rotation, Refractive index, Hemolytic activity, swelling index, Foaming index,
Astringency.
4.5 DRUG ADMINISTRATION
4.5.1 DRUG ABSORPTION
Absorption is the process by which a drug enters the bloodstream without being chemically
altered or the movement of a drug from its site of application into the blood or lymphatic system.
The rate of absorption
There are a number of factors which influence the rate of absorption and these include;
- Types of transport
- The physicochemical properties of the drug
- Protein binding
- Routes of administration
- Dosage forms
- Circulation at the site of absorption
Concentration of the drug
The rate at which a drug reaches it site of action depends on:
 Absorption - involves the passage of the drug from its site of administration into the
blood
 Distribution - involves the delivery of the drug to the tissues
Mechanisms of solute transport across membranes

33 | P a g e
- Passive diffusion
- Filtration and bulk flow
- Endocytosis
- Ion-pairing
- Active transport.

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5.0 CHAPTER FIVE: THE WRITHING GROUP PROJECT
5.1 PREPARATION OF CHENOPODIUM OPOLIFOLIUM (OMWETANGO)
Theory and introduction
Animal welfare regulations and accreditation guidelines require the use of sedatives, analgesics
or anesthetics for procedures which “may cause more than momentary or slight pain or distress.
Additionally, untreated pain increases catecholamine secretion and causes stress, which
introduces experimental variation by impairing wound healing and immune function. This
experiment was designed to see how local medicines can reduce pain in laboratory animals since
it’s their right not feel pain.
Procedure
The sample (Omwetango) 343.2g was weighed
70% Ethanol 5l was measured and then mixed with the sample for maceration to take place
The set up was left to stand for a week in a dark room
The extract was separated into two petri dishes and were then evaporated. The evaporation
expelled about litres of solvent.
The extract was put then put in an oven for efficient evaporation.
The extract was then weighed to get the percentage yield.
Procedure on dosing the mice
A total of ≅ 18ml 4.5ml for each group were used
Mice were separated and isolated into different test groups
Each mice was administered appropriate dose according to their weight (injected
introperitoneally)
5 minutes were allowed to elapse
The mice were then observed for 15 minutes and the number of writhes counted for each mice. A
writhe is indicated by stretching of the abdomen with simultaneous stretching of the hind limb.

35 | P a g e
%inhibitor =
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑟𝑖𝑡ℎ𝑒𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 −𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑟𝑖𝑡ℎ𝑒𝑠 𝑖𝑛 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑟𝑖𝑡ℎ𝑒𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
x100%
Preparation of the stock solution
The extract 15.00g was then weighed on the filter paper
The extract 15.00g was dissolved in 50ml of distilled water
The filter paper was weighed before and after dissolving the extract when dry since the sample
was sticky to ensure efficiency and effective extract.

36 | P a g e
Results
Weight of paper + Omwetango, g 395.80
Weight of empty paper, g 52.60
Weight of Omwetango, g 343.20
Weight of filter paper + the extract, g 16.60
Weight of empty filter paper, g 1.60
Weight of extract, g 15.00
Weight of residue + filter paper after drying,
g
3.20
Weight of dissolved extract, g 11.80
Experimental design and dosage level
Weight of rats and their groups
Groups Group 1 Group 2 Group 3 Group 4
No. 1 2 3 4 5 6 7 8 9 10 11 12
Weight,
g
37.7 22.6 28.5 39.3 37.8 27.2 34.8 33.4 32.6 33.0 34.0 26.7
Dosage level and treatment of results
13.4g was dissolved in 50ml of water
This means the extract is 11.8g/50ml
It was given as 11800mg containing 50ml
: Prepared stock is 236mg/ml

37 | P a g e
The extract was dissolved in 10ml for each mice
Hence the available extract was: 23.6mg/ml
STANDARD DOSE OF MICE
1g dosage level is 100mg/kg
1000g of animal takes 100mg of drug
37.7g of animal would take
37.7
100
𝑥100𝑔
= 3.77g of drug
Stock solution: 23.6g/ml of Omwetango extract
23.6g of animal takes 1ml
1
23.6
𝑥3.77𝑚𝑙
= 0.16ml/37.7g animal.
Preparation of ibuprofen
Weight of a tablet: 200mg
The tablet of ibuprofen was 200mg was crushed into powder
The powder was then dissolved in 10ml of distilled water.
Treated as:
200mg of animal takes 10ml
10
200
𝑥37.7𝑚𝑙
= 0.19ml/37.7g animal.
Discussion
Conclusion

38 | P a g e
Professional oversight and careful clinical examination is necessary to properly treat pain and
avoid toxicity or possible interference with experimental data.(according to Lee-Parritz, D… )
Local anesthetics can provide very effective pain control when used in the skin, in wound
margins and around sensory nerves. The effective use of local nerve blocks requires some
knowledge of regional anatomy and practice of the administration technique.
6.0 GENERAL CONCLUSION
The period of my training at NCRI enhanced the development of essential skills and knowledge
in herbal medicine quality control and food processing. It also helped me appreciate the various
challenges in the working environment. It was a great opportunity in acquiring practical skills
based on the theories I had learnt from class and a series of practicals held in university
laboratories. It also enabled me to see what goes on outside university and training institutions.
This was all possible due to good reputation Makerere University has and the willingness of the
authority staff to give us all the available information and skills. The training helped me increase
my relations.

39 | P a g e
REFERENCES
1. Omujal Francis, “Presentation about Organization structure of NCRI “. Natural
chemotherapeutic research institute, Wandegeya.
2. Taber, R. I. (1974). Predictive value of analgesic assays in mice and rats. Advances in
Biochemical Psychopharmacology 8,191-211.
3. Flecknell PA, Roughan JV, Stewart R. (1999) Use of oral buprenorphine
(‘buprenorphine jello’) for postoperative analgesia in rats - a clinical trial. Laboratory
Animals 33: 169-74.
4. Centre for Australian national biodiversity research, plant collection procedures, retrieved
on 31st July, 2013; http://www.anbg.gov.au/cpbr/herbarium.
5. Guidelines for assessment of herbal medicines. WHO Expert Committee on Specifi
cations for Pharmaceutical Preparations. Thirty-fourth report. Geneva, World Health
Organization, 1996 (WHO Technical Report Series, No. 863) Annex 11.
6. Bagul MS, Rajani M (2006) Phytochemical evaluation of Chyavanprash. In: Govil
N,Singh VK, Arunachalam C (eds) Recent Progress in Medicinal Plants – Search for
Natural Drugs, vol 13. Stadium, Houston, p 251
7. International Journal of Biodiversity and Conservation Vol. 4(3), pp. 101-112, March
2012 also at Available online at
http://www.academicjournals.org/IJBC
8. Barnes J, Anderson LA, Phillipson JD (2007). Herbal medicine. 3rd Edition,
Pharmaceutical Press, London. pp 1-23.
9. Bauer R (1998). Quality criteria and standardization of phytopharmaceuticals: Can
acceptable drug standards be achieved? Drug Inform. J., 32: 101–110.

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10. Grant GJ, Piskoun B, Lin A, Bansinath M. (2000) An in vivo method for the quantitative
evaluation of local anesthetics. Journal of Pharmacological and Toxicological Methods
42: 69-77.
11. Arun Rasheed, Roja C. Formulation, standardization and pharmacological evaluation of a
poly herbal traditional remedy- Ashwagandharishtam. Oriental Pharmacy and
Experimental medicine. 2012, 51-58.
12. Dabeka, R.W., Conacher, H.B., Lawrence, J.F., Newsome, W.H., McKenzie, A., Wagner,
H.P.,
Chadha, R.K. and Pepper, K. (2002) ‘Survey of bottled drinking waters sold in Canada
for
chlorate, bromide, bromate, lead, cadmium and other trace elements’, Food Additives and
Contaminants, Vol. 19, pp.721–732.

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