Figure 1: Images of electrophoresis gels showing: the integrity of the extracted genomic DNA. A 1 Kb DNA Ladder marker (Invitrogen, Carlsbad, USA) (A) was used and the absence of amplicons was verified using specific primers for BPV-2 (B), BPV-4 (C) and BPV-1 (D). The images show only the presence of amplicons consistent with the positive controls. Marker used 100 bp DNA Ladder (Invitrogen, Carlbad, USA) in figures B, C and D

Figure 2: Statistical graph comparing the score values of the treated samples in relation to the positive control, in which the C-, Saponins, DMSO and H groups showed significant differences with the comparative group, not showing a mutagenic character

Figure 3: Microscopic analysis of the Micronucleus Test in blood cells A) Lymphocytes without the presence of a micronucleus; B) Lymphocyte with micronucleus

Figure 4: Statistical graph comparing the score values of the treated samples in relation to the positive control, in which only the C- and DMSO groups showed significant differences with the comparative group

Figure 5: Comparative histogram of the percentages of micronuclei counted among the samples with the 8 different treatments in Vero cells

Figure 6: Statistical graph comparing the means with standard deviation of viable cells of the treated samples in relation to the negative control, in which only the H and L1 + H groups did not show significant differences with the comparative group

Figure 7: Dot plot graph of the data analyzed using the BD Accuri C6 software for Vero cells treated according to Table 3 based on the average percentage of live cells, using the two-way ANOVA test followed by the test Tukey post-hoc (P <0.05) using GraphPad Prism software version 5 (GraphPad Software, Inc.)

Figure 8: Statistical graph of the data analyzed using the BD Accuri C6 software for vero cells treated according to table 3 in comparison to cells without treatment (C-). Based on the average percentage of cells distributed in each phase of the cell cycle and standard deviation, the two-way ANOVA test was performed followed by the Bonferroni post-hoc test (p <0.05) using the Graphpad prism software version 5 (GrandPad Software , Inc.)

Figure 9: Chromatographic analysis of the sigma Standard saponin extract (A) and the Saponin extract under test (B), in a UFLC Shimatzu system, model Proeminence. 21 fractions were eluted, which were collected and selected 13 (indicated by arrow). The material was fractioned in a Jupiter C18 semi- preparative reverse phase column (250 mm x 10 mm, Phenomenex) with a linear gradient from 0 to 80% acetonitrile in acidified water for 60 minutes, in a flow of 2mL / min. Absorbance was monitored at 225 and 280 nm

Figure 10: Deconvolution of the spectra of the fractions of pattern nº05 and nº09. The images were obtained through analysis using the Mass Analyzer 1.03 software. Through the deconvolution of the ions (m / z), the molecular masses of 740.09 Da for fraction nº05 and 740.17 Da for fraction nº09 of the standard

Figure 11: Deconvolution of the spectra of the fractions of sample nº06 and nº 10. The images obtained through analysis by Mass Analyzer 1.03 software.
Through the deconvolution of the ions (m / z), the molecular masses of 740.15 Da for fraction No. 06 and 740.14 Da for fraction No. 10 of the standard

Primer

 

Sequence (5’-3’)

Gene
target

Expected size
of amplicon (pb)

 

BPV-2

 

 

 

GTTATACCACCCAAAGAAGACCCT
CTGGTTGCAACAGCTCTCTTTCTC

 

L2

 

164

BPV-4

 

GCTGACCTTCCAGTCTTAAT
CAGTTTCAATCTCCTCTTCA

E7

170

Degenerate Nucleotides: W - A ou T, Y – C ou T, M – C ou A, R – A ou G, K – T ou G, V – A, C ou G, H – A, C ou T, D – C, G ou T, I – A, C ou T.
Table 1:Sequences of primers for diagnosis

 

Specific Primers

 

Time

Temp.

Start

3 min

94ºC

Denaturation

50 sec

94ºC

Ringing

1 min

60ºC

Extension

1 min

72ºC

Cycles

35

Final extension

5 min

72ºC

Table 2:PCR reaction parameters using the different pairs of primers

              Abbreviations

               Tested Drugs

                  Concentration

C-

-

-

C+

Ciclofosfamida         (in PBS)

50ug/mL

L1

L1 of BPV-1 (in PBS)

1ug/mL

Al(OH)3

Aluminum Hydroxide

10uL/mL

L1+ Al(OH)3

L1 of BPV-1
Recombinant (in PBS)+
Aluminum Hydroxide

                1ug/mL + 10uL/mL

Saponin

Aqueous Extract of
Agave sisalana (in PBS and DMSO 2% )

50ug/mL

L1+Saponin

L1 of BPV-1
Recombinant (in PBS)+ Aqueous Extract of Agave
sisalana (in PBS and DMSO 2%)

                  1ug/mL +50gL/mL

DMSO

DMSO 2%+PBS

10uL/mL

Table 3:Drugs Tested and Work Concentrations

      Samples

 

        ng/µL

      R (260/230)

 

     R (260/280)

1

 

17

1, 07

 

1, 03

2

 

27

0, 93

 

1, 08

3

 

27

0, 93

 

1, 08

4

 

65

1, 00

 

1, 57

5

 

70

0, 82

 

1, 39

6

 

65

1, 06

 

1, 58

7

 

43

0, 83

 

1, 70

8

 

37

1, 32

 

1, 26

9

 

80

1, 90

 

1, 64

10

 

26

3, 15

 

1, 49

Table 4:Spectrophotometer determination of the DNA concentration of the collected blood samples and the different ratios at different wavelength.

Sample

 

Class 0

 

Class 1

 

Class 2

 

Score

 

Negative control (blood
cells w ith no substance

 

 

537

 

 

126

 

 

131

 

 

398

 

Positive control
50µg/m L cycl

 

 

25

 

 

278

 

 

500

 

 

1278

 

Experim ental group 1
(1 µg / ml of recL1)

 

 

414

 

 

173

 

 

213

 

 

599

 

Experim ental group 2
(1 µg / m L of rec L1 + 50ug / m L of Saponins

 

 

 

369

 

 

 

237

 

 

 

194

 

 

 

625

 

Experim ental group 3
(10uL / m L of Al(OH)3

 

 

 

505

 

 

 

167

 

 

 

129

 

 

 

425

 

Experim ental group 4
(50ug / m L Saponins

 

 

513

 

 

128

 

 

159

 

 

446

 

Experim ental group 5
(1 µg / ml of rec L1 + 10uL / ml of Al(OH)3

 

 

 

 

490

 

 

 

 

32

 

 

 

 

278

 

 

 

 

588

 

Contol Group (DMSO +
PBS (2%)

 

 

512

 

 

178

 

 

110

 

 

398

Number of nucleoids observed by class (0 - no damage, 1 - intermediate damage and 2 - maximum damage), and the respective score value that shows the damage index - obtained from the sum of the product of the number of nucleoids (N) by the respective value of class (C), according to the formula: Σ= (NxC0) + (NxC1) + (NxC2). Total of 8 samples.
Cycl= cyclophosphamide. Rec= recombinante


Table 5:: Results and scores obtained in the analysis of mutagenic potential by comet assay from 8 sample.

Table 6: Dunn's post-hoc test statistical values obtained using the GraphPad Prism software version 5

                Sample

Class 0

Class 1

 

Class 2

 

Score

Negative control
Only Vero cells

140

05

 

03

 

13

Positive control (50 µg /
m L cycle

17

40

 

93

 

226

Experimental group 1
1 µg / m L of rec L1

61

37

 

53

 

141

Experimental group 2
(1 µg / ml of rec L1 + 50ug / ml of Saponins)

63

29

 

58

 

145

 

Experimental group 3
(10uL / m L of Al(OH)3

61

33

 

53

 

138

 

Experimental group 4
(50ug / m L Saponins)

33

54

 

63

 

180

 

Experimental group 5
       (1 µg / ml of rec L1 + 10uL / ml of Al(OH)3

 

60

 

41

 

 

59

 

 

159

 

Control Group DMSO +
PBS (2%)

 

112

 

21

 

 

19

 

 

59

Table 7:Number of nucleoids observed by class (0 - no damage, 1 - intermediate damage and 2 - maximum damage), and the respective score value that shows the damage index - obtained from the sum of the product of the number of nucleoids (N) by the respective value of class (C), according to the formula: Σ = (NxC0) + (NxC1) + (NxC2)
Table 8: Drugs Tested and Work Concentrations

Samples

Nº Micronuclei

                      MNr0

C+

691

                      0, 691

                C-

77

                      0, 077

         Al(OH)3

148

                       0, 148

L1+ Al(OH)3

176

                       0, 176

              L1

106

                       0, 106

           Saponin

226

                       0, 226

L1+Saponin

266

                       0, 266

            DMSO

113

                       0, 113

Table 9:Number of micronuclei found per slide and calculated frequency

 

 

 

 

 

Sample

 

   Class 0

 Class 1

      Class 2

           Score

 

Negative Control

 

338

26

18

64

Positive Control
50 µg/mL de cycl

 

69

40

191

422

Control Group DMSO +
PBS 2%

 

150

111

39

189

Experimental Group
Standard (Sample 1)

 

123

48

119

286

Experimental Group
Standard (Sample 2)

 

143

76

82

240

Experimental Group
Standard (Sample 8)

 

180

102

19

140

Experimental Group
Standard (Sample 9)

 

204

21

74

214

Experimental              Group
Standard (Sample 10)

 

 

85

20

60

140

Experimental              Group
Standard Sample 5

 

 

271

72

57

286

Experimental              Group
Standard Sample 4

 

 

141

98

59

216

Experimental                 Group
AEHAS Sample 2

 

91

87

122

331

Experimental                 Group
AEHAS (Sample 6)

 

157

28

115

258

Experimental                 Group
AEHAS (Sample 7)

 

77

88

135

358

Experimental                 Group
AEHAS (Sample 8)

 

65

99

133

365

Experimental                 Group
AEHAS (Sample 10)

 

121

50

130

310

Experimental                 Group
AEHAS (Sample 11)

 

199

29

72

173

Table 10: Number of nucleoids observed by class (0 - no damage, 1 - intermediate damage and 2 - maximum damage), and the respective score value that shows the damage index - obtained from the sum of the product of the number of nucleoids (N) by the respective value of class (C), according to the formula: Σ = (NxC0) + (NxC1) + (NxC2). From 3 samples

Negative Control

Score: 64

Positive Control

Score: 422

DMSO

Score: 189

Standard nº Pico

Retencion time (min)

score

1

28,13-30,54

286

2

30,54-31,66

240

4

32,64-34,13

216

5

34,13-35,22

286

8

38,28-39,28

140

9

39,28-40,84

214

10

40,84-42,57

140

EHAS nº Pico

Retencion time (min)

Score

2

30,17-32,29

331

6

34,90-35,11

258

7

35,11-37,14

358

8

37,14-38,58

365

10

39,47-41,42

310

11

41,42-42,04

173

Table 11: Comparison between standard and sample in relation to retention time and genotoxic activity scores of the comet assay