PART II

LABORATORY TECHNIQUES AND

SCIENTIFIC TESTS IN LINE WITH
THE S.I.P.
 SCREENING,
SEPARATION AND
ANALYSIS FOR PLANT
CONSTITUENTS
 INTRODUCTION
Phytochemicals
They are the chemicals that present naturally in
plants.
Phytochemicals play a vital role against number
of diseases such as asthma, arthritis, cancer etc.
unlike pharmaceutical chemicals these
phytochemicals do not have any side
effects.
These can also be considered
as man- friendly medicines.
INTRODUCTION
Natural substances
From plants, microorganisms, animals,- etc. (totally
obtained from nature).
Semisynthetic substances
These are drugs that are manufactured by partial
synthesis.
Synthetic substances
These are drugs which are manufactured
by total synthesis (i.e. complete synthetic
process or processes).

Wagner, H., Bladt, S. & Zgainski, E. M, Plant Drug Analysis

THE PHYTOCHEMICAL SCREENING
PHYTOCHEMICAL SCREENING
To carry phytochemical screening the following points must
be fulfilled:
Selection of promising plant materials.
Proper collection of selected plants.
Authentication of plant material.
Drying of plant materials.
Grinding of the dried plants.

Richard J. P. Cannnell, Natural Product Isolation

PHYTOCHEMICAL SCREENING
To carry phytochemical screening the following
points must be fulfilled:
Garbling of the dried plants
Packing, storage and preservation
Extraction and fractionation of constituents.
Methods of separation and purification.
Methods of identification of isolated
compounds

Richard J. P. Cannnell, Natural Product Isolation

1. SELECTION OF PROMISING PLANT MATERIALS

Before investing time, effort and money in phytochemical

screening it is very important to select a promising plant.
The choice of promising plant depends upon the
following:
A plant which have a biological activity.
A plant used in folk medicine.
A plant which show a particular toxicities.

Richard J. P. Cannnell, Natural Product Isolation

2. PROPER COLLECTION OF SELECTED PLANTS
Drug may be collected from:
1- Wild plants. 2- Cultivated plants.
Wild plant Cultivated plant
Disadvantage Advantage
1- Scattered in large or Present in limited area.
unlimited area
2- Difficult to reach Easy to reach
3- The collector must be highly The collector must not be
skilled botanists skillful person
4- Deficiency may occur due to Continuous supply
continuous collection
Richard J. P. Cannnell, Natural Product Isolation
3. AUTHENTICATION OF PLANT MATERIAL
This may be confirmed by:
Establishing the identity by a taxonomy experts.
Collection of a common species in their expected
habitat by a field botanist.
By comparing the collecting plant
with a voucher specimen
( herbarium sheet)

Richard J. P. Cannnell, Natural Product Isolation

 4. DRYING OF PLANT MATERIALS
Aim of drying:
Ease of transport.
Ease of grinding
Inhibit the growth of microorganisms.
Preservative of active constituents.
Drying is done in:
Shade and in sunlight (Natural drying).
Hot air drying or by freeze-drying
(Artificial drying).
5- Grinding of the dried plants
After complete drying of plants they have to be powdered
well for further analysis
6- Garbling of the dried plants
7- Packing, storage and preservation

Richard J. P. Cannnell, Natural Product Isolation

8- EXTRACTION AND FRACTIONATION OF CONSTITUENTS

There is no general (universal) method for the extraction of

plant materials.
The precise mode of extraction depends on:
1- The texture of the plant material.
2- The water content of the plant material.
3- The type of substances to be
extracted or nature of active
constituents.
PLANT EXTRACTION
PLANT EXTRACTION
 Gathering of crushed Air-dried and
Carica papaya and P. powdered then
nigrum seeds afterwards underwent
commercially. rotary evaporation.

Recording results and

comparing them from the
varied set of
concentrations.

48 hours exposure of 20 Rearing of third instar

third instar larvae at larvae at laboratory
varied concentrations. condition.
 METHODOLOGY

Extraction of the
Cucurbita maxima Get Pre-tests of control
duch seeds and experimental pigs

Modified Wisconsin Sugar

Floatation Technique

Day 1 exposure (30ml of the

extract) and get the fecal
samples of the pig

Day 3 exposure (50ml of the Day 2 exposure (40ml of the

extract) and get the fecal extract) and get the fecal
samples of the pig samples of the pig
Nanoparticle Test
ALLIUM TEST
 Albert
Levan
in1938
influence and
mutual action
between Allium establishing
genotoxic
Test
genotoxicity
substances
and genetic
material
(CA) is
established in
the root tip
cells
 TEST ORGANISMS
1. Equal-sized bulbs are chosen from a
population of the common onion
Allium cepa and grown in a test
liquid. The onions should have a size
of 15-22 mm and a weight of 2-4 g.

2. The test material is easily stored

under dry conditions by +10-2OoC
 TEST ORGANISMS
3. Prior to test start, the outer scales of the
bulbs and the brownish bottom plate should
be removed, the ring of root primordia being
left intact. Onions are grown in control
medium for first 24 hour

4. Put the individual onions onto 15-ml-Falcon

tubes, filled with control medium. The base of
the onion must reach the medium surface.
Cover the tube-stand with aluminium to keep
the onion roots in dark during growth.
Incubate them at 251 C in cultivator with
light cycle.
 ROOT GROWTH INHIBITION TEST

1. The Allium cepa test consists in

obtaining onion bulbs cultivated
without the application of
herbicides or fungicides
2. After obtaining the bulbs, they
should be scraped at the root to
promote the emergence of new
roots
 ROOT GROWTH INHIBITION TEST

3. To set-up the experiment allowing rootlets to grow, all bulbs should be

placed initially in a small 50 mL plastic cup (Figure 1), containing distilled
or tap water (being that it is potable), for approximately 03 to 04 days
so rootlets can emerge
 MACERATION OF THE ROOT TIPS AND
PREPARATION FOR MICROSCOPY
1. After exposition exclude the plant
with the poorest root growth. Use
the rest 5 plants per sample for the
microscopy.
2. Cut 5 root tips per plant at a length
of 10 mm and place them into 10-
ml-glass tube with 2 ml acetic
acid/HCl solution
 MACERATION OF THE ROOT TIPS AND
PREPARATION FOR MICROSCOPY
1. After exposition exclude the plant
with the poorest root growth. Use
the rest 5 plants per sample for the
microscopy.
2. Cut 5 root tips per plant at a length
of 10 mm and place them into 10-
ml-glass tube with 2 ml acetic
acid/HCl solution
3. Heat the root tips for 5 minutes at
50 oC. Hereby, the root cells will
become fixated and macerated.
 MACERATION OF THE ROOT TIPS AND
PREPARATION FOR MICROSCOPY

4. Thereafter, place the root tips on a

microscope-slide on a black
background and cut off the terminal
tips (1-2 mm) for further preparation.

5. Remove the rest of root material

and liquid from the slide.
 MACERATION OF THE ROOT TIPS AND
PREPARATION FOR MICROSCOPY
6. Add 2 drops of orcein solution and mix
it with the roots properly by stirring and
knocking with a stick of stainless steel
(stirring spattle).
7. Place a cover slip on the root cells. The
staining procedure takes about 5-10 min.
8. After that squash the cells by placing
to layers of filtrate paper on the cover
glass and pressing slightly down with
thumb. Follow the microscopy
immediately or fix the cover slip to the
slide with clear nail varnish. Such a slide
can be kept in the freezer fresh up to 2
months.
 MACERATION OF THE ROOT TIPS AND
PREPARATION FOR MICROSCOPY
8. After that squash the cells by
placing to layers of filtrate paper on
the cover glass and pressing slightly
down with thumb.
9. Follow the microscopy immediately
or fix the cover slip to the slide with
clear nail varnish. Such a slide can be
kept in the freezer fresh up to 2
months.
Parameters can be Used to Estimate the Cytotoxicity, Genotoxicity, and
Mutagenicity of Environmental Pollutants

Root Shape
This parameter is observable after 3-5 days of treatment that show
swelling, bending and discoloration of the root tips or roots.
 Root Length and EC50 Determination
Root growth decrease over 45% indicates the presence of toxic nature of
substances having sublethal effects on plants.
EC50 (Half Maximal Effective Concentration): it is the effective concentration of a
chemical producing 50% of the total effect.

Growth inhibition of Allium cepa roots exposed to Growth inhibition of Allium cepa roots exposed
textile effluent. to paint effluent.
Mean ( SD) root length of A. cepa exposed to different concentrations of industrial effluents.
 Mitotic Index (M.I)
Mitotic Index (MI) is a parameter of cytotoxicity in studies of
environmental biomonitoring.
The cytotoxic level can be determined by the decrease rate of mitotic index. A mitotic
index decrease below 22% of negative control causes lethal effects on test organism
while a decrease below 50% has sublethal effect (cytotoxic limit value).

Several investigators have used MI as an endpoint for the evaluation

of genotoxicity or antigenotoxicity of different chemical treatments.
X 100
Total Number of Cells
in the Field of View =
93

Total Number of Cells

undergoing Cell
Division = 63

M.I. = 67.74
Chromosomal Aberrations (CAs)
CAs are characterized by change in either total
number of chromosomes or in chromosomal
structure which occur as a result of the
exposure of chemical treatment.

Diploid metaphase chromosome from the root

cells of the onion (Allium cepa L.), containing
2n of 16 (2n=16).
Damaged chromosomes: abreak in centromere, bdouble break
chromatide, csingle break chromatide, dgap chromatide.
DEVELOPMENTAL TOXICITY
OR TERATOGENESIS ASSAYS
 Duck Embryo
1

Prepare the extract

2

Obtain 1-day old fertilized duck eggs from

3 duck farm
 Assign eggs to control and treatment
4 groups

Scratch shell with knife with ridges

5

Inject extract ( 3 does) with and without

6 retinoic acid to treatment groups
 Inject vehicle to negative control
7

Inject retinoic acid ( 1ug/ml) to positive

8 control

Incubate at 370C, 60% relative humidity

9
 Observe daily mortality
10

Open up 3 eggs every five days

11

Observe chorioallantoic membrane blood

12 vessel
13
Observe vitelline blood vessels

14
Take pictures

12
Dissect out the embryo
END OF PART 2
RESEARCH DESIGN
 RESEARCH DESIGN

A plan or strategy for conducting the research

A complete sequence of steps to be
undertaken to ensure that the appropriate
data will be obtained in a way which permits
an objective analysis leading to valid
inference with respect to the stated problem
 IMPORTANCE OF THE RESEARCH
DESIGN
It serves as a guide for the conduct of the
research for the conduct of the research work.
It allows a gain of maximum information
relevant to the problem at minimum cost
It helps control variance., thus, increasing the
validity of procedures and results.
Variance = sq.rt. of standard deviation
no. of samples
Controlling variance being able to explain or
account for variance caused by variables
being studied
Variance difference in values obtained from
the different samples
 CHARACTERISTICS OF A GOOD RESEARCH
DESIGN
Research should not be do-able but should
yield results that can be interpreted with
confidence.
1. Freedom from bias
- data vary on the basis of random
fluctuation
- bias may be eliminated by random
assignment of individuals or random sampling
 CHARACTERISTICS OF A GOOD RESEARCH
DESIGN
2. Freedom from confounding
- two or more variables are confounded if
their effects cannot be separated
3. Statistical precision for testing hypothesis
 THE FOLLOWING CONSIDERATION ARE
ALWAYS PRESENT IN A RESEARCH DESIGN
Randomization the assignment of experimental
units to the treatment or vice versa by chance
Eliminates bias
Assures validity of the statistical test of
significance
 Replication the repetition of the basic
experiment; done to provide an estimate of
variation among observations or units treated
alike to assess significance of observed
difference.

Number of replicates needed is based on:

1. Degree of precision needed
2. Degree of homogeneity of samples
3. Number of treatment in the study
 FACTORS TO BE CONSIDERED IN
YOUR RESEARCH DESIGN:
Experimental unit e.g. test organism
Sampling Methods
Treatment/Treatment combinations
Control
Measurement
Statistical design for testing
 IN PLANNING FOR YOUR RESEARCH
DESIGN, FIRST CONSIDER THE
FOLLOWING:
What type of research are you going to
conduct based on approach?
Descriptive Research?
Experimental research?
 ARE YOU GOING TO CONDUCT A
DESCRIPTIVE RESEARCH?
Example of descriptive researches :
Taxonomic Studies
e.g. Taxonomic classification of flowering plants in
Guimaras Island
Community Structure Studies
e.g. Density and species abundance of
seagrassess in Talin Bay, Batangas
 Descriptive-Normative Survey
E.g. Population characteristics of a particular
species of bird located in Mt. Makiling
Data collected would include: body wt.
wing span, beak length, age, color pattern
Statistical analysis would involve mean,
standard deviation, shape of the distribution
curve
 EXPERIMENTAL RESEARCH
VARIABLES ARE MANIPULATED
Independent variable manipulated (cause)
Dependent variable result /effect
Example: The effect of organic and inorganic fertilizer on the
growth of pechay
Independent variable: type of fertilizer
Dependent variable : growth of pechay ( e.g. weight of
plant; width of leaves etc.)
Extraneous variable other variables which cannot be
controlled and may affect the results ( e.g. air temperature,
humidity etc.)
 ARE YOU GOING TO CONDUCT AN
EXPERIMENTAL RESEARCH?
Experimental Designs:
A.Simple Experimental Design ( Two-group Design)
a.1 Comparing Two groups of Equal Sizes
b.E.g. Effects of cassava feeds and commercial
feeds on the weight of tilapia fries after two
months of culture
 Experimental Designs:
A. Simple Experimental Design ( Two-group Design)
a. 1 Comparing Two groups of Equal Sizes
E.g. two types of fertilizers on crop yield
Crop yield (bu/acre)
Plot New fertilizer Old Fertlizer
1 67.4 60.6
2 72.8 66.6
3 68.4 64.9
4 66.0 61.8
5 70.0 61.7
6 69.6 67.2
7 67.2 62.4
8 68.9 62.4
9 62.6 56.7
N= 9
 Weight (gm) of femaie rats
High protein diet Low protein diet
134 70
146 118
Experimental Designs:
104 101
A. Comparing two groups of 119 85
unequal sizes 124 107
E.g. Effect of high protein diet and 161 132
low protein diet on the weights of 107 94
female rats after two months 83 n= 7
113
129
97
123
n= 12
 Paired design: Self-Pairing
E.g. Effect of Sargassum tablet supplement on weight (gm) of
white mice after two weeks.

Weight (gm) of white mice (gm)

R Before treatment After treatment
1 84.5 89.1
2 86.9 89.8
3 87.0 90.9
4 85.2 89.9
5 86.7 90.1
6 87.4 91.5
N= 6 Mean = 86.03 Mean= 90.20
 Complex Design: Comparing more than two groups
e.g. Anti-fungal of plant extract of fusarium with fungi
Diameter zone of inhibition (mm) of the different treatments on
Fusarium with Fungi

R (-) (+) Aloe Calamansi Tomato

control control vera
( distilled (Beniate)
water)
1 0 20 8 18 20
2 0 20 12 24 22
3 0 22 7 21 16
N= 3
 CRITERIA OF A WELL-DESIGNED
EXPERIMENT
Adequate experimental control (effect can be detected)
Lack artificiality ( Real World)
Basis of comparison ( control- experimental group)
Adequate information from the data ( stat. generated)
Uncontaminated data ( reflect good and poor effect)
No confounding of relevant variables ( adequate
experimental control)
THE SCIENTIFIC METHOD
ACTIVITY
 PARAMETRIC TEST:
COMPARISON OF TWO
MEANS
T test is a parametric statistical test used to
establish the significant difference between the
means of two samples

Paired Samples t-test

- Compares the mean scores of the same group
before and after treatment is given or the mean
scores of the same group with two different
treatments.
 EXAMPLE
Title: Treatment of industrial Wastewater using Kappaphycus
alvarezii (Red Algae)
Total 1 2 3 4 5
dissolved
Solid (ppt)
TDS 2.45 2.23 1.97 2.20 2.05
before
treatment
(X)
TDS after 1.02 0.93 0.88 0.64 1.21
treatment
(y)
1. State the Null and Alternative Hypothesis
Ho- There is no significant difference in the amount of total dissolved solids of industrial
wastewater before and after treatment with Kappapyycus alvarezii
H1 :There is a significant difference in the amount of total dissolved solids of industrial
wasterwater before and after treatment with Kappaphycus alvarezii

2. Determine the level of Significance (a) a=0.05

* We are 95% confident that we will not reject the hypothesis given that is true.
3. Determine the test statistics: paired samples t-test
4. Determine the degrees of freedom (df) df=n-1
df=n-1=5-1=4
Determine the critical region (c.r)
Critical region an area in the normal distribution curve wherein if the computed
values fall within it, the null hypothesis is rejected; check the table for t values in the
corresponding d.f. and a.
+/- 4,0.5 = +/- 2.776 if computed t<-2.776 or t>2.776, null hypothesis is rejected
 EXAMPLE
Title: Treatment of industrial Wastewater using Kappaphycus
alvarezii (Red Algae)
Total 1 2 3 4 5
dissolved
Solid (ppt)
TDS 2.45 2.23 1.97 2.20 2.05
before
treatment
(X)
TDS after 1.02 0.93 0.88 0.64 1.21
treatment
(y)
5. Compute the test statistics

N=5 X Y d d2
1 2.45 1.02 1.43 2.0449
2 2.45 1.02 1.30 1.69
3 2.45 1.02 1.09 1.1881
4 2.45 1.02 1.56 2.4336
5 2.45 1.02 0.84 0.7056
Total 6.22 8.0622
(!)
a. Solve for d d=X-Y
b. Solve for d and d2
c. Solve for d. d= (d/n=6.22/5=1.244
d. Solve for standard deviation
SD= 0.284833
e. Solve for t-value
t= d/sd/n = 1.244/0.284833 5 = 9.766
6. Make statistical decision
Since the computed value 9.766 in the critical
region, i.e computed t=9.766>2.776, the null hypothesis is
rejected. Alternative hypothesis is accepted.
7. Make a conclusion
There is a significant difference in the total dissolved
solids of industrial wastewater before and after treatment
with Kappaphycus alvarezii
 T-TEST FOR INDEPENDENT
MEANS/SAMPLES
Compares the mean scores of two different or independent
groups.
Example: Survival rate of Native Clam (Clam venerupis) in Two
Estuaries in Manila bay.
Survival rate of Native Clam (Clam venerupis) in Two Estuaries in Manila
bay.
Area 1 2 3 4 5 6 7

1 97.9 81.8 40.5 99.3 86.2 74.3 76.0

2 78.9 67.1 73.0 69.1 96.4 86.8 66.6

1. State the Null and Alternative Hypothesis
Ho- There is no significant difference Survival rate of Native Clam
(Clam venerupis) in Two Estuaries in Manila bay
2. Determine the level of Significance (a) a=0.05
* We are 95% confident that we will not reject the hypothesis
given that is true.
3. Determine the test statistics: T-test for independent means
4. Determine the degrees of freedom (df)
df=n1+n2-2 d.f. = 7+7-2 =12
Determine the critical region (c.r)
c.r. +/- t = 0.05, 12 = +/-2.179 t < -2.179 or t>2.179
5. Compute the test statistics
Area 1 ( N=7) (S1 = SD of area 1)
X1 X1- x1 (X1- x1)2

1 97.9 18.47 341.1409

2 81.8 2.37 5.6169
3 40.5 -38.93 1515.5449
4 99.3 19.87 394. 8169
5 86.2 6.77 45.8329
6 74.3 -5.13 26.3169
7 76.0 -3.43 11.7649
Total (!) 556 2341.0343

X1=79.43 S1=19.75278166
5. Compute the test statistics
Area 2 ( N=7) ( S2 = SD of Area 2)
X2 X2- x2 (X2- x2)2

1 78.9 2.06 4.2436

2 67.1 -9.74 94.8676
3 73.0 -3.84 14.7456
4 69.1 -7.74 59.9076
5 96.4 19.56 382.5936
6 86.8 9.96 99.2016
7 66.6 -10.24 104.8576
Total (!) 537.9 760.4172

X1=76.84 S1=19.75278166
a. Solve for mean and SD of each group
b. Solve for variance (Sp)
sp = s12(n1-1) + (s22(n2-1)
n1 + n2 -1
sp = 16. 076511366
c. Solve for t value
t = 0.301399073
6. Statistical decision: Since the computed value t is less
than 2.179, it does not fall within the critical region, there
is no sufficient evidence to reject the null hypothesis.

7. Conclusion:
There is no significant difference in the survival rate
of native clam (Native Clam (Clam venerupis) in Two
Estuaries in Manila bay.
CONDUCTING AND WRITING
S.I.P IS NOT EASY.

The story of my friend.

Romans 12:2
Do not conform to the pattern of this
world, but be transformed by the renewing
of your mind. Then you will be able to test
and approved what Gods will is-his good,
pleasing and perfect will.
 BOTTOM LINE:
CHANGE YOUR PERSPECTIVE.
 If we only have the right
perspectives, we will have the right
view in conducting and writing
Science Investigatory Project
 ASK YOURSELF?
High school public school students who are less in
opportunity join Intel Science Fair for Science
Investigatory Project and represent our country
abroad. Knowing that you are more privilege students
than them; can you do the same thing and represent
the Philippines all over the world?
Thank you for
listening and
participating

Prof. Glen R. Mangali

Colegio de San Juan de Letran
La Salle College Antipolo
Philippine Normal University