ffTraining-Workshop on

RESEARCH LABORATORY
TECHNIQUES
for High School and Elementary
Science Research

14-15 SEPTEMBER 2019, UST-ANGELICUM COLLEGE, QUEZON CITY JASON B. ALBARO

Research
Don't know what to do
Whenever you are near
Don't know what to say
My heart is floating in tears
When you pass by I could fly
Research
I finally found someone, that knocks me off
my feet
I finally found the one, that makes me feel
complete
We started over coffee, we started out as
friends
It's funny how from simple things, the best
things begin..
Research
'Til I met you
I never knew what love was
'Til I met you
This feeling seems to grow more
Every day
I love you more each day…
Research
Research is the hope of our
future…
 ISEF RULES
*Use of Vertebrate Animal
*Use of Human and Animal Tissue Samples
*Use of Human as Respondents
*Potentially Hazardous Biological Agents
*Risk Assessment
*Qualified Scientist
GUESS THE WORD!
APCELRTNIOO
PERCOLATION
ENRCATIAMO
MACERATION
IONUSNFI INFUSION
XSOETHL REXOTACNIT
SOXHLET EXTRACTION
NECDOITOC
DECOCTION
NINSOTOACI
SONICATION
 OBJECTIVES
•To enumerate the different preparations made
before extractions
•To explain the methods of extracting plant
materials
•To get acquainted with researches related to
plant extraction PRECAUTIONARY MEASURES
•Be careful in handling chemicals
•Wear proper attire/clothes when
performing laboratory activities
PLANT EXTRACTION
Plant extraction is the separation of active
portions of plant tissues using selective solvents
through standard procedures. The basic parameters
influencing the quality of an extract .
•Plant part used as starting material
•Solvent used for extraction
• Extraction procedure
MEDICINAL PLANTS ARE THE RICHEST BIO-RESOURCE
•drugs of traditional systems of medicine,
•modern medicines,
•nutraceuticals,
•food supplements,
•folk medicines,
•pharmaceutical intermediates,
•chemical entities for synthetic drugs.
CHOICE OF SOLVENTS
•Successful determination of biologically active
compounds depends on the type of solvent used in
the extraction procedure

Property of a good solvent in plant extraction

•Low toxicity
•Ease of evaporation at low heat
•Promotion of rapid physiologic absorption of the
extract •Preservative action
The factors affecting the choice of solvent

•Quantity of phytochemicals to be extracted

•Rate of extraction
•Diversity of different inhibitory compounds extracted
•Ease of subsequent handling of the extracts
•Toxicity of the solvent in the bioassay process
•Potential health hazard of the extractants
STEPS INVOLVED IN PLANT
COLLECTION
STEPS INVOLVED IN PLANT
COLLECTION Collection of plants
Plants under consideration may be collected either from
wild forests or from herbariums. When plants are collected
from wild, there is a risk that they have been incorrectly
identified. The major advantage of wildlife plants is that they
will not contain any pesticides. After the plants are collected
from wild or from herbarium they have to be processed for
cleaning in order to prevent the deterioration of
phytochemicals present in plants.
STEPS INVOLVED IN PLANT
COLLECTION Cleaning of Plants

After plants collection they have to be cleaned

properly. The cleaning process may involve the following
steps. Cleaning, washing, peeling or stripping leaves
from stems. Cleaning has to be done by hands in order to
get better results.
STEPS INVOLVED IN PLANT
COLLECTION
Drying

The main purpose of drying is to remove the water

content from plants so that the plants can be stored. Plants
have to be dried immediately as soon as the plants
collection or this will lead to spoilage of plant materials. The
drying consists of two methods. Drying can be done either
by natural process or by artificial process.
STEPS INVOLVED IN PLANT

Drying COLLECTION

A. Natural Process
Natural process includes sun- drying. Sometimes plants
are placed on drying frames or on stands, to be air-dried in
barns or sheds. But this may take few weeks for complete
drying. The time depends on temperature and humidity.
STEPS INVOLVED IN PLANT
Drying COLLECTION

Artificial Drying
Artificial drying can be done with the help of artificial driers. This
process will reduce the drying time to several hours or minutes. The
common method that is followed in drying medicinal plants is warm-
air drying. In this process plants are placed in the plates of drier on
which warm air is blown. This method is mainly applicable to fragile
flower and leaves and this requires large number of workers since
loading and unloading of plants has to be done manually.
STEPS INVOLVED IN PLANT
COLLECTION
Powdering

After complete drying of plants they have to be

powdered well for further analysis
METHODS OF EXTRACTION
METHODS OF EXTRACTION
Plant Tissue Homogenization
Plant tissue homogenization in solvent has been widely
used by researchers. Dried or wet, fresh plant parts are
grinded in a blender to fine particles, put in a certain quantity
of solvent and shaken vigorously for 5 - 10 min or left for 24 h
after which the extract is filtered. The filtrate then may be
dried under reduced pressure and re-dissolved in the solvent
to determine the concentration. Some researchers however
centrifuged the filtrate for clarification of the extract
 METHODS OF EXTRACTION
Serial Exhaustive Extraction
It is another common method of extraction which involves
successive extraction with solvents of increasing polarity from a non
polar (hexane) to a more polar solvent (methanol) to ensure that a
wide polarity range of compounds could be extracted. Some
researchers employ soxhlet extraction of dried plant material
using organic solvent. This method cannot be used for thermolabile
compounds as prolonged heating may lead to degradation of
compounds
 METHODS OF EXTRACTION
Soxhlet Extraction
Soxhlet extraction is only required where the desired compound
has a limited solubility in a solvent, and the impurity is insoluble in that
solvent. If the desired compound has a high solubility in a solvent then
a simple filtration can be used to separate the compound from the
insoluble substance. The advantage of this system is that instead of
many portions of warm solvent being passed through the sample, just
one batch of solvent is recycled. This method cannot be used for
thermolabile compounds as prolonged heating may lead to
degradation of compounds
 METHODS OF EXTRACTION
Soxhlet Extraction
ADVANTAGES
1. We use a small amount of solvent
2. It is a closed apparatus so that it is used for dangerous
organic solvents.
3. It can be used for the extraction of different active
constituents by changing the polarity of the solvent.
4. It can be used for the extraction of active constituent
decomposed by direct heat.
 METHODS OF EXTRACTION
Maceration
In maceration (for fluid extract), whole or
coarsely powdered plant- drug is kept in contact
with the solvent in a stoppered container for a
defined period with frequent agitation until soluble
matter is dissolved. This method is best suitable for
use in case of the thermolabile drugs.
 METHODS OF EXTRACTION
Decoction
This method is used for the extraction of the
water soluble and heat stable constituents from
crude drug by boiling it in water for 15 minutes,
cooling, straining andpassing sufficient cold water
through the drug toproduce the required volume
.
 METHODS OF EXTRACTION
Infusion
It is a dilute solution of the readily soluble
components of the crude drugs. Fresh infusions are
prepared by macerating the solids for a short period
of time with either cold or boiling water .
 METHODS OF EXTRACTION
Digestion

This is a kind of maceration in which gentle heat

is applied during the maceration extraction process.
It is used when moderately elevated temperature
is not objectionable and the solvent efficiency of the
menstrum is increased thereby .
 METHODS OF
EXTRACTION
Percolation
This is a kind of maceration in which gentle heat is applied during the
maceration extraction process. It is used when moderately elevated
temperature is not objectionable and the solvent efficiency of the
menstrum is increased thereby mass is packed and the top of the
percolator is closed. Additional menstrum is added to form a shallow layer
above the mass, and the mixture is allowed to macerate in the closed
percolator for 24 h. The outlet of the percolator then is opened and the liquid
contained therein is allowed to drip slowly. Additional menstrum is added
as required, until the percolate measures about three- quarters of the
required volume of the finished product. The marc is then pressed and the
expressed liquid is added to the percolate. Sufficient menstrum is added to
produce the required volume, and the mixed liquid is clarified by filtration
or by standing followed by decanting .
 METHODS OF EXTRACTION
Sonication
The procedure involves the use of ultrasound with
frequencies ranging from 20 kHz to 2000 kHz; this increases the
permeability of cell walls and produces cavitation. Although the
process is useful in some cases, like extraction of rauwolfi a root,
its large-scale application is limited due to the higher costs. One
disadvantage of the procedure is the occasional but known
deleterious effect of ultrasound energy (more than 20 kHz) on the
active constituents of medicinal plants through formation of free
radicals and consequently undesirable changes in the drug
molecules
 METHODS OF EXTRACTION

Distillation
In this method we use a special apparatus which is
called Clavenger it is used mainly for the extraction of
volatile compound, e.g. orange peels has been used for
the extraction of orange oil. and clove has been used for
the extraction of clove oil .
SIMPLE Distillation METHODS OF EXTRACTION
 Distillation is a
common operation in
many laboratories
for the purpose
of
separating and/or
purifying
components of a
liquid mixture of varying
boiling points.
METHODS OF EXTRACTION
FRACTIONAL Distillation
Fractional distillation is the
separation of a mixture into its
component parts, or fractions.
Chemical compounds are
separated by heating them to a
temperature at which one or
more fractions of the mixture will
vaporize. It uses distillation to
fractionate.
 SAMPLE
RESEARCH/STUDY
 SAMPLE
RESEARCH/
STUDY
 SAMPLE
RESEARCH/
STUDY
SAMPLE RESEARCH/ STUDY
ARATILES (Muntigua calabura L.)LEAVES
EXTRACT WOUND HEALING BULM
 SAMPLE
RESEARCH/
STUDY
 REFERENCES:
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Research in Chemical Science (IJARCS)
BegumS,HassanSI,SiddiquiBS,ShaheenF,GhayurMN,etal.(2002)Triterpenoidsfrom the leaves of Psidium guajava. Phytochemistry 61:
399-403.
Boca Raton: Taylor & Francis. Cowan, M.M. (1999): Plant products as antimicrobial agents. Clinical Microbiology Reviews. 12(4): 564 – 582.
Chapatwala, K.D.; Boykin, M.A. & Rajanna, B. (1982). Effects of intraperitoneally injected cadmium on renal and hepatic glycogenic
enzymes in rats. Drug Chemistry and Toxicology. 5:305-317.
Chen, F.W.; Shieh, P., Kuo, D. & Hsieh, C. (2006). Evaluation of the antioxidant activity of Ruellia tuberosa. Food Chemistry. 94: 14-18. Choi,
S.W.; Friso, S. & Editors. (2006).
Daffre, S.;Bulet, P., Spisni, A., Ehret-sabatier, L., Rodrigues, EG. &Travassos, L.R. (2008). Bioactive natural peptides. In: Atta-ur-Rahman (Ed.)
Studies in Natural Products Chemistry, Vol. 35. Elsevier. pp 597- 691.

Das K, Tiwari RKS, Shrivastava DK. Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and
future trends. Journal of Medicinal Plants Research 2010; 4(2): 104-111.

Evans.W.C, “Treaseand Evans Pharmacognosy”, Harcourt Brace and company. Asia pvt. Ltd.Singapore, 1997.

Finar,I.L, “Stereo Chemistry and the Chemistry of Natural Products” Longman, VOL.2, 1986.

Gahan. P.B. “Plant Histochemistry and Cytochemistry: An Introduction. Academic Press, Florida, U.S.A, 1984..
Handa SS, Khanuja SPS, Longo G, Rakesh DD. Extraction Technologies for Medicinal and Aromatic Plants. International centre for science
and high technology, Trieste, 2008, 21-25.
Harborne.J.B, “Phytochemical Methods: A Guide To Modern Techniques of Plant Analysis.” 2nd Edition, Chapmann and Hall Publishers,
London, 1998.

REFERENCES:
James.E, K.Robbers, m.speedie and E.Varro Tyler, Pharmacognosy and pharmaco Biotechnology, Williams and Willkins, New York, U.S.A,
1996.
Kim HB, Bang HS, Lee HW, Seuk YS, Sung GB (1999) Chemical characteristics of mulberry syncarp. Korean J Med. Crop Sci 47: 32063209.
Kokate,C.K, “Practical pharmacognosy” 4 th edition, Vallabh Prakashan Publication, New Delhi, India, 1999.

Mace.M.D, “Histichemical localization of phenols in healthy and diseased tomato roots”, Phytopathology, 1963, 16:915-925. N.

Raaman, “Phytochemical Techniques “, New India Publishing Agency, New Delhi, ISBN- 81-89422-30-8.

Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods
and future trends. African Journal of Biotechnology 2008; 7 (12): 1797-1806.
Nikhal SB, Dambe PA, Ghongade DB, Goupale DC. Hydroalcoholic extraction of Mangifera indica (leaves) by Soxhletion. International
Journal of Pharmaceutical Sciences 2010; 2 (1): 30-32.
Osman AM, Younes ME, Sheta AE (1974) Triterpenoids of the leaves of Psidium guajava. Phytochem 13: 2015-2016.

Rasch. E. and H.Swift, “Micro Photometric Analysis of the Cytochemical million eaction”, J.Histochem.Cytochem, 1960, 8:4-17.
Remington JP. Remington: The science and practice of pharmacy, 21st edition, Lippincott Williams & Wilkins, 773-774.
Wagner.H, „Pharmazeutische Biologic”, 5 th Edition, AUFI.15 BN 3-437-20 498-X, 1993.
Whistler.R.L. and J.N. BeMiller, „Industrial Gums; Polysaccharides and their Derivatives, Academic Press, London, 1993.
Yasuma. A and Ichikawa. “Ninhydrin-Schiff and alloxan- Schiff staining. A new histochemical staining methods for proteins”, J. Lab clin
Med, 1953, 41:296-299.

ACTIVITY ON PLANT EXTRACTION

Group 1 to 3: percolation
Group 4 to 6: INFUSION
Group 7 to 9: DECOCTION
Group 10 to 12: Maceration
Using your worksheets, you need to make a proposed project that
would involve the process assigned to your group.
 PRESENTATION
2 groups from each cluster will share their
work/output
THANK YOU!