Part 2 3 Conducting SIP Laboratory Technique Glen Mangali

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 Carica papaya and P. nigrum seeds commercially.

Air-dried and powdered then afterwards underwent rotary evaporation.

Recording results and comparing them from the varied set of concentrations.

48 hours exposure of 20 third instar larvae at varied concentrations.

Rearing of third instar larvae at laboratory condition.

METHODOLOGY Extraction of the Cucurbita maxima duch seeds

Get Pre-tests of control 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 extract) and get the fecal samples of the pig

Day 2 exposure (40ml of the extract) and get the fecal samples of the pig

Nanoparticle Test

ALLIUM TEST

Albert Levan in1938 influence and mutual action between genotoxic substances and genetic material

Allium Test (CA) is established in the root tip cells

establishing genotoxicity

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 25±1 °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 10ml-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 10ml-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 textile effluent.

Growth inhibition of Allium cepa roots exposed 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: a–break in centromere, b–double break chromatide, c–single break chromatide, d–gap chromatide.

DEVELOPMENTAL TOXICITY OR TERATOGENESIS ASSAYS

1 2 3

•Duck Embryo •Prepare the extract •Obtain 1-day old fertilized duck eggs from duck farm

4 5 6

•Assign eggs to control and treatment groups •Scratch shell with knife with ridges •Inject extract ( 3 does) with and without retinoic acid to treatment groups

7 8 9

•Inject vehicle to negative control •Inject retinoic acid ( 1ug/ml) to positive control •Incubate at 370C, 60% relative humidity

10 11 12

•Observe daily mortality •Open up 3 eggs every five days •Observe chorioallantoic membrane blood vessel

13 14 12

•Observe vitelline blood vessels •Take pictures •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

• Experimental Designs: A. Comparing two groups of unequal sizes E.g. Effect of high protein diet and low protein diet on the weights of female rats after two months

134

70

146

118

104

101

119

85

124

107

161

132

107

94

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

1 2 3 N= 3

(-) (+) control control ( distilled (Beniate) water) 0 20 0 20 0 22

Aloe vera

Calamansi

Tomato

8 12 7

18 24 21

20 22 16

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 t2.776, 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 1 2 3 4 5 Total (!∑)

X 2.45 2.45 2.45 2.45 2.45

Y 1.02 1.02 1.02 1.02 1.02

d 1.43 1.30 1.09 1.56 0.84 6.22

d2 2.0449 1.69 1.1881 2.4336 0.7056 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 2 3 4 5 6 7 Total (!∑)

97.9 81.8 40.5 99.3 86.2 74.3 76.0 556

18.47 2.37 -38.93 19.87 6.77 -5.13 -3.43

341.1409 5.6169 1515.5449 394. 8169 45.8329 26.3169 11.7649 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 2 3 4 5 6 7 Total (!∑)

78.9 67.1 73.0 69.1 96.4 86.8 66.6 537.9

2.06 -9.74 -3.84 -7.74 19.56 9.96 -10.24

4.2436 94.8676 14.7456 59.9076 382.5936 99.2016 104.8576 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 God’s 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