APPENDICES

71


Appendix 1
Certification and Other Documents




72








73







74








75


Appendix 2
Raw Data



Table 17. Brine Shrimp survival within 24 hours
Sugod-sugod
leaf extract
Log
Concentration(x
)

I II III % Mortality
Probit
value
(y)
10 mcg/ml 1 10 9 10 3.33% (3%) 3.12
100 mcg/ ml 2 8 10 10 6.67% (7%) 3.52
1000 mcg/ml 3 7 8 7
26.67%
(27%)
4.39
Control 10 10 10


Linear Regression Results
Total number: 3
Slope (b): 0.635
y-intercept (a): 2.40667
Regression equation (y); 2.407 + 0.635x
Y= 2.407 + 0.635x
5- 2.407+ 0.635x
2.593
= x
0.635
x = 4. 083

LC
50
= (shift log/ anti-log) 4.083
=12, 118.94 mcg/ml


76


Table 18. Zone of inhibition of S. aureus
Treatments Trail I Trial II Trial III Mean (mm)
Sugod-sugod leaf
extract 250mg/ml
5.1 6.2 8.2 6.5
Sugod-sugod leaf
extract 500mg/ml
12.8 11.6 15.9 13.43
Sugod-sugod leaf
extract 1000mg/ml
20.1 19.2 21.8 20.37
Positive control
Ciprofloxacin (Xipro)
75.80 51.65 75.8 67.75
Negative control
(distilled water)
0 0 0 0

Table 19. Zone of inhibition of S. pyogenes
Treatments Trail I Trial II Trial III Mean (mm)
Sugod-sugod leaf
extract 250mg/ml
6.20 3.90 2.90 4.33
Sugod-sugod leaf
extract 500mg/ml
12.20 7.70 6.90 8.93
Sugod-sugod leaf
extract 1000mg/ml
19.9 20.7 19.3 19.97
Positive control
Ciprofloxacin (Xipro)
50.1 75.8 51.8 59.23
Negative control
(distilled water)
0 0 0 0

Table 20. Zone of inhibition of E. coli
Treatments Trail I Trial II Trial III Mean (mm)
Sugod-sugod leaf
extract 250mg/ml
10.50 9.50 9.10 9.70
Sugod-sugod leaf
extract 500mg/ml
11.20 11.10 9.90 10.73
Sugod-sugod leaf
extract 1000mg/ml
12.50 12.80 11.70 12.33
Positive control
Ciprofloxacin (Xipro)
13.60 15.90 14.70 14.73
Negative control
(distilled water)
0 0 0 0



77




Table 21. Group Statistics of EC
50
values of Sugod-sugod and ascorbic acid
GROUP N Mean Std. Deviation Std. Error
Mean
EC50VALUES SUGOD-
SUGOD
ASCORBIC ACID
3
3
89.000
48.0533
1.97198
.33247
1.13852
.19195

Table 22. Independent Samples Test for EC
50
values (Levenes Test for
Equality of Variances)
Levenes Test for Equality of
Variances
F Sig.
EC50VALUES Equal variances
assumed
Equal variances not
Assumed
10.189 .033

Table 23. Independent Samples Testt-test for Equality of Means
t-test for Equality of Means
T Df Sig. (2-
failed)
Mean Difference
EC50VALUES
Equal variances
assumed

Equal variances not
Assumed
35.464
35.464
4
2.114
.000
.001
40.94667
40.94667

Table 24. Independent Samples Testt-test for Equality of Means
t-test for Equality of Means
Std. Error
Difference
95% Confidence Interval of
the Difference
Lower Upper
EC50VALUES Equal variances
assumed
Equal variances not
Assumed
1.15459
1.15459
37.74101
36.22618
44.15232
45.66716




78


One-way ANOVA
Table 25. Descriptives of bacteria
N Mean Std. Deviation Std. Error
STAPHYLOCOCCUS 250mg
500mg
1000mg
positive control
negative control
Total
3
3
3
3
3
15
6.500
13.4333
20.3667
67.7500
.0000
21.6100
1.57162
2.21886
1.32035
13.94301
.00000
25.47395
.90738
1.28106
.76231
8.05000
.00000
6.57735
STREPTOCOCCUS 250mg
500mg
1000mg
positive control
negative control
Total
3
3
3
3
3
15
4.3333
8.9333
19.9667
59.2333
.0000
18.4933
1.69214
2.85715
.70238
14.37231
.00000
22.87174
.97696
1.64958
.40552
8.29786
.00000
5.90546
ECOLI 250mg
500mg
1000mg
positive control
negative control
Total
3
3
3
3
3
15
3.7000
4.7333
6.3333
8.7333
.0000
4,700
.72111
.72342
.56862
1.15036
.00000
3.06431
.41633
.41767
.32830
.66416
.00000
.79120

Table 26. Descriptives of bacteria
95% Confidence
Interval for Mean


Minimum


Maxim
um
Lower
Bound
Upper
Bound
STAPHYLOCOCCUS 250mg
500mg
1000mg
positive control
negative control
Total
2.5959
7.9214
17.0867
33.1136
.0000
7.5030
10.4041
18.9453
23.6466
102.3864
.0000
35.7170
5.10
11.60
19.20
51.65
.00
.00
8.20
15.90
21.80
75.80
.00
75.80
STREPTOCOCCUS 250mg
500mg
1000mg
positive control
negative control
Total
.1298
1.8358
18.2219
23.5305
.0000
5.8274
8.5368
16.0309
21.7115
94.9361
.0000
31.1593
2.90
6.90
19.30
50.10
.00
.00
6.20
12.20
20.70
75.80
.00
75.80
ECOLI 250mg
500mg
1000mg
positive control
negative control
Total
7.9087
8.9363
10.9208
11.8757
.0000
6.5882
11.4913
12.5304
13.7459
17.5910
.0000
12.4188
9.10
9.90
11.70
13.60
.00
.00
10.50
11.20
12.80
15.90
.00
15.90


79



Table 27. Analysis of variance of bacteria
Sum of Squares df Mea
n
Squa
re
STAPHYLOCOCCUS Between Groups
Within Groups
Total
8677.823
407.088
9084.911
4
10
14
2169.
456
40.70
9
STREPTOCOCCUS Between Groups
Within Groups
Total
6887.463
436.167
7323.629
4
10
14
1721.
866
43.61
7
ECOLI Between Groups
Within Groups
Total
126.080
5.380
131.460
4
10
14
31.52
0
.538


Table 28. Analysis of variance of bacteria
F Sig.
STAPHYLOCOCCUS Between
Groups
Within Groups
Total
53.292 .000
STREPTOCOCCUS Between
Groups Within
Groups
Total
39.477 .000
ECOLI Between
Groups
Within Groups
Total
177.361 .000

Table 29. Duncans multiple range test of S. aureus

GROUP

N
Subset for alpha = 0.05
1 2 3 4
negative control
250mg
500mg
1000mg
positive control
Sig.
3
3
3
3
3

.0000
6.5000



.241

6.5000
13.4333


.213


13.4333
20.3667

.213




67.750
0
1,000
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 3,000STREPTOCOCCUS
80



Table 30. Duncans multiple range test of S. pyogenes

GROUP

N
Subset for alpha = 0.05
1 2 3
negative control
250mg
500mg
1000mg
positive control
Sig.
3

3
3
3
3
.000

4.3333
8.9333

.14



8.9333
19.9667
.066



59.2333

1,000
Means for groups in homogeneous subsets are displayed
a. Uses Harmonic Mean Sample Size = 3,000.




Table 31. Duncans multiple range test of E. coli

GROUP

N
Subset for alpha = 0.05
1 2 3 4
negative control
250mg
500mg
1000mg
positive control
Sig.
3
3
3
3
3

.0000




1,000



9.7000
10.7333


.115



12.3333

1,000




14.7333
1,000

Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 3,000.









81


Appendix 3
Preparation For The Difeerent Tests

Preparation of Dragendorffs reagent
In an empty container 0.85g bismuth(III) nitrate was dissolved in a mixture of 10
milligrams acetic acid and 40 milligrams water. In another container 8grams potassium
iodide was dissolved in 20 milligrams water. Equal parts of both solutions were mixed.
The resulting mixture was stored in a dark bottle for a long time at room temperature.
The reagent was mixed by preparing 1mL stock solution with 2 milligrams of acetic acid
and 10 milligrams water. Another 1mL of the filtrated sample was taken and tested with
Mayers reagent.
Preparation of Mayers reagent1.
Four grams mercuric iodide was dissolved in 60 milligrams water. The resulting
mixture was poured into a solution of 5.0grams potassium iodide that was dissolved in
10 milligrams water. Enough water was added to make 100milligrams.The positive
results for Dragendorffs reagent indicate by an orange precipitateand reagent a white
precipitate for Myers reagent.

Artificial sea water
The 3.8grams of rock salt was dissolved in a 100 mL of distilled water.

Food for the shrimp
A suspension of 3mg of dried yeast (Red Star) was prepared in 3 mL artificial
water.


82


Preparation of Culture Media
Preparation of Mueller-Hinton Agar.Suspend 38 g of the medium in one liter of
purified water. Heat with frequent agitation and boil for one minute to completely dissolve
the medium and autoclave for 15 minutes at 21C. Cool to room temperature. Pour
cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface to give
uniform depth. Allow to cool to room temperature.













83


Appendix D
Documentation





Figure 8.Sugod-sugod leaves







84



Figure 9. Phytochemical Screening for Alkaloids



Figure10 . Phytochemical Screening of Flavonoids

85



Figure 11. Phytochemical Screening for Saponins


Figure 12. Phytochemical Screening for Tannins



86



Trials I II III (250 mg/ml)

Trials I II III (500 mg/ml)


Trials I II III (1000 mg/ml)


Trials I II III ( Ciprofloxacin- positive control)


Trials I II III (distilled water- negative control)

Figure 13. Zone of Inhibition on Staphylococcus aureusby the Different Treatment
87



Trials I II III (250 mg/ml)


Trials I II III (500 mg/ml)


Trials I II III (1000 mg/ml)


Trials I II III ( Ciprofloxacin- positive control)


Trials I II III (distilled water- negative control)

Figure 14. Zone of Inhibition on Streptococcus pyogenes by the Different Treatment
88



Trials I II III (250 mg/ml)


Trials I II III (500 mg/ml)


Trials I II III (1000 mg/ml)


Trials I II III ( Ciprofloxacin- positive control)


Trials I II III (distilled water- negative control)

Figure 15. Zone of Inhibition on E. coli by the Different Treatments
89














Figure 16. Different Concentrations of Brine shrimp Toxicity Assay