Evaluation of mutagenic property of poly herbal formulation of annona squamosa, zingiber officinalis and triticum aestivum plant extracts by bacterial reverse mutation test

Rajesh R; Jagadeesh Singh S D; Channabasava R

doi:https://doi.org/10.26452/ijrps.v10i4.1657

Evaluation of mutagenic property of poly herbal formulation of annona squamosa, zingiber officinalis and triticum aestivum plant extracts by bacterial reverse mutation test

Rajesh R ✉ ¹ , Jagadeesh Singh S D ² , Channabasava R ³

1 Department of Biotechnology, Tumkur University, Tumkur- 572106, Karnataka, India, +91-9902317306

2 Department of Pharmacognosy and Biotechnology, East Point Research Academy, Bangalore, Karnataka, India

3 Department of Toxicology, Liveon Biolabs Private Limited, Tumakuru, Karnataka, India

Abstract

Cancer is the second deadliest cause among all ailments even after the development of modern research and technologies. This leads in search of alternative sources of treating cancer. Herbal formulations were considered to be the rich sources of treating agents for most of the ailments from agelong days. Bacterial Reverse Mutation method of evaluation is one among the preliminary phase incorporated to assess the mutagenic potential of herbal formulations. The selected Polyherbal Formulation (PF3) of Annona Squamosa, Zingiber Officinalis, and Triticum Aestivum in 1:2:3 ratio was evaluated for its mutagenic potential. The results with method I - Plate incorporation and method II - Preincubation indicate that, the PF3 extract at a maximum dose of 5 mg/plate did not cause a positive increase in the mean number of revertant colonies per plate with any of the tester strains either in the presence (+S9) or absence of metabolic activation (-S9). The rich flavonoids and other phytoconstituents of the formulation may be responsible for antimutative properties with the possible inactivation of mutagens or by interfering in the process of mutagenisis. The higher studies were needed to be proven further to authenticate the mutagenic potentialities.

Keywords

Mutagenicity, Poly herbal, Reverse mutation, Salmonella typhimurium, Escherichia coli, Toxicity

Introduction

Cancer is considered as the second leading cause of death among all the ailments globally. It was estimated that 9.6 million deaths as in 2018, i.e., 1 among the 6 deaths is due to cancer (WHO, 2018) even after the pharma revolution.

Preparation of Positive Controls

Positive control samples were prepared as per Table 3. The biggest challenge in treating cancer is to retain the metabolic integrity of normal cells. Chemotherapeutic agents like fluorouracil cause myelo and cardiotoxicities (Macdonald, 1999; Rexroth & Scotland, 1983), doxorubicin causes cardiac, renal and myelotoxicities (Avilés, Arévila, Maqueo, & Nambo, 1993; Gibaud, Andreux, Weingarten, Renard, & Couvreur, 1994; Manil, Mahieu, & Couvreur, 1995), Cyclophosphamide cause cardio, immune and alopecic defects (Fraiser, Kanekal, & Kehrer, 1991).

Apart from the development of new treatment methods and sources, herbal formulations are the choice from an age-long period in treating long term ailments due to their nontoxic, non-accumulative, economical, and almost less side effects.

Table 1: Co-Factor Salt Solution for S9 Mix

NADP Na (4 mM)		283 mg
D-glucopse-6-phosphate (Di sodium salt) (5mM)	:	126 mg
mgCl2 (8mM)	:	146 mg
KCL (33 mM)	:	221 mg

Table 2: Test System Characterization

Sl. No.	Strain	Reversion events	Mutation	Additional/Mutation alterations	Plasmids
Salmonella typhimurium
1.	TA98	Frame shift	hisD3052	rfa mutation/uvrB deletion	pKM101
2.	TA100	Base-pair substitution	hisG46	rfa mutation/uvrB deletion	pKM101
3.	TA1535	Base-pair substitution	hisG46	rfa mutation/uvrB deletion	-
4.	TA1537	Frame shift	hisC3076	rfa mutation/uvrB deletion	-
Escherichia coli
5.	uvrA	Base-pair substitution	trpE65	uvrA deletion	-

Table 3: Preparation of Positive Controls

Sl. No.	Chemicals	Concentration (µg/plate)	Quantity	Solvent (DMSO)	Metabolic Activation (S-9)	Bacterial Strains
1.	2-Nitrofluorene	2	40 µg	2 mL	-	TA98
2.	2-Aminoanthracene	5	100 µg	2 mL	+	TA98, TA100, TA1535, E. coli WP2 uvrA
3.	Sodium Azide	1	20 µg	2 mL	-	TA100, TA1535
4.	4-nitroquinoline 1-oxide	100	2 µg	2 mL	-	E. coli WP2 uvrA
5.	9-Aminoacridine	15	300 µg	2 mL	-	TA1537

Note: The S9 fraction used in this study was prepared in-house. Standardization of metabolic activity of the S9 fraction (Batch No: S9-01-2015) was carried out using 2 mutagens -2-Aminoanthracene and Benzo-(a)-pyrene. The numbers of revertant colonies with both mutagens were with-in the acceptance range derived from the in-house historical data. Hence, in this study, only one mutagen - 2-Aminoanthracene was used as a positive control.

Table 4: Acceptance range of revertant colonies

Bacterial strains	Without S-9	With S-9
Salmonella typhimurium TA 98	20-50	20-50
Salmonella typhimurium TA 100	45-200	45-200
Salmonella typhimurium TA 1535	5-20	5-20
Salmonella typhimurium TA 1537	5-20	5-20
E. coli WP2 UVrA	10-50	10-50

Table 5: Record for Viable Count of Tester Strains

Sl. No	Name of the strain	Number of colonies in 10-7 dilutions			Mean & SD
		R1	R2	R3
1.	TA98	127	119	123	123 ± 4
2.	TA100	125	128	141	131 ± 9
3.	WP2 uvrA	116	109	122	116 ± 7
4.	TA1535	150	146	155	150 ± 5
5.	TA1537	126	124	130	120 ± 3

Table 6: Record for Preliminary Toxicity Test

Test item	Number of revertant colonies (S. TA100)
concentration	With S-9			Without S-9
(µg/plate)	Plate 1	Plate 2	Mean & SD	Plate 1	Plate 2	Mean & SD
Solvent control (DMSO)	65	67	66 ± 1.41	73	75	74 ± 1.41
50	76	72	74 ± 2.83	76	74	75 ± 1.41
100	68	69	69 ± 0.71	80	79	80 ± 0.71
200	71	68	70 ± 2.12	71	74	73 ± 2.12
400	78	72	75 ± 4.24	65	68	67 ± 2.12
833	64	65	65 ± 0.71	74	77	76 ± 2.12
1600	82	80	81 ± 1.4	76	79	78 ± 2.12
3200	70	73	72 ± 2.1	69	74	72 ± 3.54
5000	68	67	68 ± 0.7	72	70	71 ± 1.41

Note: All values are expressed as CFU Mean ± Standard Deviation, CFU: colony-forming units, DMSO= Dimethyl sulfoxide

Table 7: Individual colony counts (CFU) of method I: Plate incorporation (Without metabolic activation)

Test conc. (/plate)

Plate TA 98

Plate TA 100

Plate E coli

Plate TA 1535

Plate TA 1537

Solvent

Control (DMSO)

(0.1 mL)

1) 28

2) 22

3) 26

28) 73

29) 87

30) 77

55) 18

56) 16

57) 17

82) 10

83) 12

84) 11

109) 08

110) 10

111) 12

0.062 mg

4) 27

5) 30

6) 23

31) 81

32) 61

33) 68

58) 16

59) 20

60) 13

85) 08

86) 11

87) 09

112) 07

113) 08

114) 11

0.185 mg

7) 20

8) 25

9) 27

34) 74

35) 83

36) 80

61) 20

62) 19

63) 22

88) 10

89) 08

90) 09

115) 12

116) 09

117) 08

0.556 mg

10) 29

11) 26

12) 24

37) 86

38) 76

39) 82

64) 26

65) 28

66) 21

91) 07

92) 08

93) 12

118) 09

119) 04

120) 10

1.667 mg

13) 30

14) 31

15) 26

40) 91

41) 95

42) 88

67) 30

68) 27

69) 25

94) 13

95) 07

96) 12

121) 12

122) 11

123) 13

2.5 mg

16) 31

17) 28

18) 26

43) 82

44) 86

45) 83

70) 26

71) 28

72) 30

97) 14

98) 08

99) 09

124) 11

125) 09

126) 12

3.75 mg

19) 31

20) 33

21) 26

46) 86

47) 102

48) 95

73) 34

74) 31

75) 33

100) 16

101) 12

102) 09

127) 14

128) 11

129) 13

5 mg

22) 36

23) 33

24) 32

49) 97

50) 99

51) 99

76) 29

77) 36

78) 25

103) 16

104) 15

105) 13

130) 16

131) 09

132) 11

Positive

control

2-Nitrofluorene

Sodium azide

4-Nitroquinoline

Sodium azide

9-Aminoacridine

25) 409

26) 397

27) 435

52) 462

53) 485

54) 448

79) 185

80) 143

81) 176

106) 176

107) 180

108) 222

133) 165

134) 197

135) 182

Note: TA= Salmonella typhimurium, DMSO=Dimethyl sulfoxide, CFU=Colony Forming Units

Table 8: Individual colony counts (CFU) of method I: Plate incorporation (With metabolic activation)

Test conc. (/plate)

Plate TA 98

Plate TA 100

Plate E coli

Plate TA 1535

Plate TA 1537

Solvent

Control (DMSO)

(0.1 mL)

1) 20

2) 25

3) 23

28) 64

29) 70

30) 72

55) 20

56) 14

57) 11

82) 12

83) 10

84) 08

109) 05

110) 09

111) 08

0.062 mg

4) 23

5) 24

6) 26

31) 78

32) 84

33) 72

58) 19

59) 21

60) 15

85) 09

86) 11

87) 08

112) 06

113) 09

114) 05

0.185 mg

7) 29

8) 22

9) 26

34) 75

35) 76

36) 74

61) 23

62) 20

63) 22

88) 07

89) 05

90) 04

115) 11

116) 07

117) 09

0.556 mg

10) 25

11) 27

12) 24

37) 80

38) 76

39) 82

64) 21

65) 23

66) 20

91) 07

92) 08

93) 10

118) 11

119) 13

120) 10

1.667 mg

13) 21

14) 26

15) 27

40) 91

41) 75

42) 82

67) 22

68) 25

69) 26

94) 09

95) 05

96) 04

121) 12

122) 09

123) 08

2.5 mg

16) 23

17) 28

18) 26

43) 83

44) 86

45) 74

70) 29

71) 26

72) 28

97) 06

98) 07

99) 10

124) 04

125) 08

126) 10

3.75 mg

19) 30

20) 32

21) 27

46) 68

47) 81

48) 87

73) 22

74) 23

75) 27

100) 11

101) 13

102) 12

127) 05

128) 07

129) 11

5 mg

22) 28

23) 30

24) 29

49) 80

50) 84

51) 91

76) 26

77) 22

78) 29

103) 10

104) 09

105) 07

130) 14

131) 11

132) 09

Positive

control

2- Aminoanthracene

25) 388

26) 355

27) 371

52) 441

53) 472

54) 412

79) 150

80) 188

81) 142

106) 155

107) 172

108) 168

133) 199

134) 151

135) 170

Note: TA= Salmonella typhimurium, DMSO=Dimethyl sulfoxide, CFU=Colony Forming Units

Table 9: Summary of a method I: Plate incorporation

Test	No. of Revertants/plate (Mean of 3 plates)
concentration	Without S-9
( /plate)		TA 98	TA 100	WP2 uvrA	TA 1537	TA 1535
Solvent Control	Mean	25	79	17	10	11
DMSO (0.1 mL)	SD	3	7	1	2	1
0.062 mg	Mean	27	70	16	9	9
	SD	4	10	4	2	2
0.185 mg	Mean	24	79	20	10	9
	SD	4	5	2	2	1
0.556 mg	Mean	26	81	25	8	9
	SD	3	5	4	3	3
1.667 mg	Mean	29	91	27	12	11
	SD	3	4	3	1	3
2.5 mg	Mean	28	84	28	11	10
	SD	3	2	2	2	3
3.75 mg	Mean	30	94	33	13	12
	SD	4	8	2	2	4
5 mg	Mean	34	98	30	12	15
	SD	2	1	6	4	2
Positive control		2-Nitrofluorene	Sodium azide	4-Nitroquinoline	9-Aminoacridine	Sodium azide
	Mean	414	465	168	181	193
	SD	19	19	22	16	25

Note: All values are expressed as CFU Mean ± Standard Deviation, TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU: Colony Forming Unit

Table 10: (Contd..). Summary of a method I: Plate incorporation

Test		No. of Revertants/plate (Mean of 3 plates)
concentration		With S-9
( /plate)		TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537
Solvent Control	Mean	21	69	15	10	7
(DMSO (0.1 mL)	SD	4	4	5	2	2
0.062 mg	Mean	23	78	18	9	7
	SD	4	6	3	2	2
0.185 mg	Mean	22	75	22	5	9
	SD	7	1	2	2	2
0.556 mg	Mean	25	79	21	8	11
	SD	2	3	2	2	2
1.667 mg	Mean	25	83	24	6	10
	SD	3	8	2	3	2
2.5 mg	Mean	22	81	28	8	7
	SD	4	6	2	2	3
3.75 mg	Mean	30	79	24	12	8
	SD	3	10	3	1	3
5 mg	Mean	29	85	26	9	11
	SD	1	6	4	2	3
Positive control		2- Aminoanthracene
	Mean	371	442	160	165	173
	SD	17	30	25	9	24

Note: All values are expressed as CFU Mean ± Standard Deviation, TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU: ColonyForming Unit

Table 11: Individual colony counts (CFU) of Method-II: Pre-incubation (Without metabolic activation)

Test conc. (/plate)

Plate TA 98

Plate TA 100

Plate E coli

Plate TA 1535

Plate TA 1537

Solvent

Control

(DMSO)

(0.1 mL)

1) 21

2) 26

3) 23

28) 65

29) 71

30) 69

55) 41

56) 43

57) 46

82)15

83) 09

84) 11

109) 10

110) 16

111) 08

0.062 mg

4) 24

5) 23

6) 26

31) 64

32) 61

33) 62

58) 42

59) 44

60) 40

85) 10

86) 12

87) 09

112) 07

113) 09

114) 06

0.185 mg

7) 25

8) 30

9) 28

34) 77

35) 74

36) 78

61) 48

62) 42

63) 41

88) 13

89) 17

90) 16

115) 05

116) 08

117) 11

0.556 mg

10) 27

11) 21

12) 26

37) 73

38) 81

39) 89

64) 36

65) 44

66) 41

91) 14

92) 16

93) 15

118) 14

119) 16

120) 16

1.667 mg

13) 23

14) 27

15) 25

40) 86

41) 80

42) 82

67) 43

68) 47

69) 42

94) 14

95) 11

96) 18

121) 11

122) 16

123) 18

2.5 mg

16) 28

17) 24

18) 29

43) 75

44) 79

45) 79

70) 44

71) 45

72) 39

97) 12

98) 17

99) 19

124) 16

125) 14

126) 11

3.75 mg

19) 31

20) 29

21) 26

46) 88

47) 88

48) 82

73) 43

74) 42

75) 40

100) 15

101) 11

102) 14

127) 15

128) 12

129) 13

5 mg

22) 32

23) 30

24) 27

49) 76

50) 79

51) 84

76) 39

77) 34

78) 31

103) 18

104) 13

105) 11

130) 16

131) 19

132) 15

Positive

control

2-Nitrofluorene

Sodium azide

4-Nitroquinoline

Sodium azide

9-Aminoacridine

25) 361

26) 412

27) 398

52) 493

53) 488

54) 454

79) 154

80) 133

81) 115

106) 222

107) 239

108) 156

133) 238

134) 254

135) 196

Note: TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU=ColonyForming Units

Table 12: Individual colony counts (CFU) of Method-II: Pre-incubation (with metabolic activation)

Test conc. (/plate)

Plate TA 98

Plate TA 100

Plate E coli

Plate TA 1535

Plate TA 1537

Solvent

Control

(DMSO)

(0.1 mL)

1) 25

2) 29

3) 26

28) 60

29) 69

30) 65

55) 32

56) 30

57) 35

82) 13

83) 07

84) 10

109) 08

110) 11

111) 07

0.062 mg

4) 27

5) 25

6) 24

31) 62

32) 59

33) 60

58) 32

59) 34

60) 31

85) 09

86) 11

87) 08

112) 04

113) 07

114) 10

0.185 mg

7) 21

8) 24

9) 22

34) 82

35) 80

36) 80

61) 41

62) 40

63) 38

88) 12

89) 10

90) 17

115) 11

116) 10

117) 13

0.556 mg

10) 25

11) 26

12) 24

37) 80

38) 75

39) 72

64) 32

65) 29

66) 38

91) 11

92) 13

93) 16

118) 12

119) 13

120) 16

1.667 mg

13) 20

14) 26

15) 28

40) 79

41) 77

42) 78

67) 35

68) 31

69) 36

94) 11

95) 15

96) 16

121) 09

122) 12

123) 14

2.5 mg

16) 26

17) 21

18) 30

43) 78

44) 72

45) 73

70) 29

71) 32

72) 31

97) 15

98) 12

99) 10

124) 16

125) 18

126) 13

3.75 mg

19) 31

20) 28

21) 26

46) 74

47) 77

48) 82

73) 34

74) 32

75) 30

100) 16

101) 11

102) 12

127) 09

128) 12

129) 14

5 mg

22) 25

23) 26

24) 24

49) 80

50) 85

51) 82

76) 32

77) 29

78) 38

103) 11

104) 13

105) 16

130) 12

131) 13

132) 16

Positive

control

2- Aminoanthracene

25) 351

26) 402

27) 392

52) 487

53) 476

54) 446

79) 152

80) 129

81) 112

106) 219

107) 228

108) 247

133) 226

134) 243

135) 188

Note: TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU=Colony Forming Units

Table 13: Summary of Method-II: Preincubation

Test concentration (/plate)		No. of Revertants/plate (Mean of 3 plates)
		Without S-9
		TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537
Solvent Control	Mean	20	68	43	11	12
(DMSO) (0.1 mL)	SD	4	3	3	4	3
0.062 mg	Mean	24	62	42	10	7
0.062 mg	SD	2	2	2	2	2
0.185 mg	Mean	28	76	44	15	8
0.185 mg	SD	3	2	4	2	3
0.556 mg	Mean	25	81	40	15	15
0.556 mg	SD	3	8	4	1	1
1.667 mg	Mean	23	83	44	14	15
1.667 mg	SD	5	3	3	4	4
2.5 mg	Mean	23	78	43	16	14
2.5 mg	SD	6	2	3	4	3
3.75 mg	Mean	29	86	42	13	13
3.75 mg	SD	3	3	2	2	2
5 mg	Mean	33	80	35	14	17
5 mg	SD	4	4	4	4	2
Positive control		2-Nitrofluorene	Sodium azide	4-Nitroquinoline	Sodium azide	9-Aminoacridine
	Mean	390	478	134	205. 7	229
	SD	26	21	20	44	30

Note: All values are expressed as CFU Mean ± Standard Deviation, TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU: Colony Forming Units

Table 14: (Contd.).Summary of Method-II: Preincubation

Test concentration ( /plate)		No. of Revertants/plate (Mean of 3 plates)
		With S-9
		TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537
Solvent Control	Mean	17	65	32	10	9
(DMSO) (0.1 mL)	SD	2	5	3	3	2
0.062 mg	Mean	20	60	32	9	7
0.062 mg	SD	4	2	2	2	3
0.185 mg	Mean	19	81	40	13	11
0.185 mg	SD	3	1	2	4	2
0.556 mg	Mean	25	76	33	13	14
0.556 mg	SD	1	4	5	3	2
1.667 mg	Mean	25	78	34	14	12
1.667 mg	SD	4	1	3	3	3
2.5 mg	Mean	26	74	31	12	16
2.5 mg	SD	5	3	2	3	3
3.75 mg	Mean	28	78	32	13	12
3.75 mg	SD	3	4	2	3	3
5 mg	Mean	25	82	33	13	14
5 mg	SD	1	3	5	3	2
Positive control		2- Aminoanthracene
	Mean	382	470	131	231	219
	SD	27	21	20	14	28

Note: All values are expressed as CFU Mean ± Standard Deviation, TA= Salmonella typhimurium, DMSO= Dimethyl sulfoxide, CFU: Colony Forming Units

The present study was aimed to determine the mutagenic potential of Polyherbal Formulation using (OECD, 1997) Bacterial Reverse Mutation Test.

Materials and Methods

Preparation of Plant Extracts

The three Annona Squamosa, Zingiber Officinalis and Triticum Aestivum plant samples collected were chopped into small pieces, shade dried and grounded using hammer type milling machine at the Department of Pharmacology, East Point Pharmacy College, Bangalore.

The powdered materials in 1:2:3 gram equivalent ratio were transferred into and extracted in the Soxhlet extractor using methanol for 72 h.

The extracts were filtered through a Whatman filter paper No. 42 (125 mm) and concentrated using a rotary evaporator with the water bath set at 40°C, then dried in a desiccator over anhydrous CuSO4.

The powdered residue was transferred into vials and stored at 4°C in airtight vials before analysis.

Metabolic Activation System

Tester strains used in the study do not possess the phase I and phase II metabolic capability to convert poromutagens into mutagenic metabolites.

In order to overcome from this major drawback, an exogenous metabolic activation system was used in the form of a mammalian microsomal enzyme activation mixture (Liver extracts obtained from Rats pre-treated with Phenobarbital/β-nepthoflavone).

Tester strains were exposed to Polyherbal PF3 extract dilutions in the presence of a metabolic activation system, i.e., cofactor supplemented post-mitochondrial fraction (S9).

In a house, a prepared S9 mixture was used in all phases of the study.

Co-Factor Salt Solution for S9 Mix

All co-factors are dissolved in 0.2 µ filter-sterilized 90 mL of Sodium Phosphate Buffer (0.2M, pH 7.4). S9 mix (Table 1 ) was prepared by mixing 10 mL of S9 homogenate with 90 mL of the Co-factor solution.

The complete S9 mix was freshly mixed just before the treatment, and the S9 mix was kept in an ice bath throughout the process.

Contents of Plate without Metabolic Activation

2 mL top agar with 10% Histidine Biotin/Tryptophan + 0.5 mL phosphate buffer saline + 0.1 mL test item/solvent control/positive control.

Contents of Plate with Metabolic Activation

2 mL top agar with 10% Histidine Biotin/Tryptophan + 0.5 mL S9 mix + 0.1 mL test item/solvent control/positive control

Bacterial Cultures Selection Criteria

Each tester strains were inoculated in to sterile Oxoid nutrient broth no. 2 and incubated at 37°C at 150 rpm for overnight (approximately 14-16 hours) in a shaker water bath maintained at 37°c, and 60 shakes/minute.

Post incubation optical density of culture was measured using a spectrophotometer at 600 nm.

The optical density of the culture solution was adjusted to 1.0 at 600nm (~1x10⁹cfu/ mL).

Controls

Solvent Control: DMSO was used to treat the test system with and without metabolic activation.

Positive Control

Combination of positive control and tester strains used in the assay is as following,

Test System Characterization

Details of the Test system are as per Table 2.

Procedure

Preparation of Test Article Dilutions

All the test article dilutions were prepared freshly using DMSO, just prior to the treatment, and were used within 2 hrs of the preparation. The required quantity of test article was weighed and suspended inappropriate volume of DMSO to prepare the stock solution of 50 mg/ mL concentration for the Preliminary Toxicity test. The remaining treatment solutions were prepared by subsequent dilution of stock solution using DMSO.

Precipitation Test

100µl from Polyherbal PF3 extract dilutions 50 mg/ mL, 40 mg/ mL, 30 mg/ mL, 20 mg/ mL and 10 mg/ mL concentrations were mixed with 2 mL top agar separately and plated over preincubated minimal glucose agar plates in duplicates. The plates were kept at room temperature for incubation for 2 hours, and Post incubation plates were observed for the presence of precipitation. No precipitation was observed even at the highest concentration of 5 mg/plate. Hence, 5 mg/plate was selected as the highest concentration for further assay.

Preliminary Toxicity Test

100µl mL of overnight culture of tester strain Salmonella TA 100 (~10⁹ cells), 100µL from test item dilutions of 50 mg/ mL, 32 mg/ mL, 16 mg/ mL, 8 mg/ mL, 4 mg/ mL, 2 mg/ mL, 1 mg/ mL and 0.5 mg/ mL to get (5 mg, 3.2 mg, 1.6 mg, 0.8 mg, 0.4 mg 0.2 mg 0.1 mg and 0.05 mg/plate), 0.5 mL of S9 mix (+S9)/0.5 mL of phosphate buffer saline (-S9) were mixed with molten top agar containing Histidine and Biotin solution (2 mL). The mixture was mixed thoroughly by vertexing and poured over pre-incubated minimal glucose agar plates in duplicates. The test was conducted in both in the presence (+S9) and the absence of a metabolic activation system (-S9). The plates were incubated at 37±1°C for 48 hours. Post incubation, the plates were inspected for bacterial background lawn and number of spontaneous revertant colonies.

Mutagenicity Assay

The polyherbal extract was tested for mutagenicity at concentrations of 0.062 mg, 0.185 mg, 0.556 mg, 1.667 mg, 2.5 mg, 3.75 mg and 5 mg/plate in both with and without metabolic activation in two different methods as following:

Method-I: Plate Incorporation

In this method, 0.1 mL polyherbal extract dilutions/positive controls, 0.1 mL tester strain (cell count of ~1x10⁹ cells) and 0.5 mL of S9 mixture/phosphate buffer were pipetted into a tube containing 2 mL sterile top agar with Histidine-Biotin for Salmonella/Tryptophan solution for E.coli strains.

The mixture was vortexed and plated over preincubated minimal glucose agar plates in triplicates. Plates were allowed to solidify and then incubated in an incubator at 37±1°C for 64 hours.

The test was conducted both in the presence (+S9) and the absence of a metabolic activation system (-S9). After the incubation, revertant colonies in each plate were counted.

Method-II: Preincubation

In this method, 0.1 mL of polyherbal extract/positive controls, 0.1 mL of tester strains (cell count of ~1x10⁹ cells) and 0.5 mL of S9 mixture/phosphate buffer was pipette into a sterile tube containing, 2 mL of top agar with Histidine-Biotin for Salmonella/Tryptophan solution for E.coli strains.

The mixture was vortexed and plated over preincubated minimal glucose agar plates. The plates were allowed to solidify and then incubated in an incubator at 37±1°C for 72 hours.

The test was conducted in both the presence and absence of a metabolic activation system. Post incubation, the Number of revertant colonies in each plate was counted.

Acceptance Criteria

The assay is considered as valid, as the following criteria were met,

a. A concentration-related increase over the range tester and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without a metabolic activation system.

b. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

c. The mean of the spontaneous revertant colonies in negative control/solvent control founds falls within the acceptance range as derived from the in-house historical data. If the value is more than the range, it will be considered as positive.

d. Positive control chemicals should induce at least a two-fold increase in a number of revertant colonies compared to the negative control/ solvent control.

e. Small increases in apparent genotoxicity in vitro or in vivo will first be assessed for reproducibility and biological significance. Examples of results that are not considered biologically meaningful include:

f. Small increases that are statistically significant compared with the negative or solvent control values but are within the confidence intervals of the appropriate historical control values for the testing facility

g. Weak/equivocal responses that are not reproducible

h. If either of the above conditions applies, the weight of evidence indicates a lack of genotoxic potential, the test will be considered negative or the findings not biologically relevant, and no further testing will be called for.

i. Acceptance range of revertant colonies (Table 4) (as per in-house historical data).

Statistical Analysis/Data Treatment

Individual counts of the number of revertant colonies/concentration were presented for the test items with mean and Standard deviation fold induction over the baseline were calculated using the following formula

Results and Discussion

Precipitation Test

Post-incubation, no precipitation was observed even at the highest concentration of 5 mg/plate. Hence, 5 mg/plate was selected as the highest concentration for further assay.

Preliminary Toxicity Test

Doses tested in the mutagenicity assay were selected based on the results of the preliminary toxicity study conducted on the test article using tester strains Salmonella TA 100 in both the presence and absence of S9 mix. 08 doses of test article, ranging from 5 mg, 3.2 mg, 1.6 mg, 0.8 mg, 0.4 mg 0.2 mg 0.1 mg and 0.05 mg/plate were tested, and results are presented in Table 5. No cytotoxicity was observed with either tester strain in the presence or absence of S9 mix, as evidenced by no dose-related decrease in the number of revertant colonies per plate. Bacterial background lawns were observed to be normal at the highest concentration of 5 mg/plate (Table 6).

Mutagenicity Assay

Results of the Preliminary toxicity study were used to select doses tested in the mutagenicity assay. Doses tested in the mutagenicity assay with all tester strains in both the presence and absence of S9 mix were 5, 3.75, 2.5, 1.667, 0.556, 0.185, and 0.062 mg/plate.

In the method I (Plate incorporation) and method II (Pre-incubation assay), all data were with-in the acceptable range. No positive increase in the mean number of revertant colonies per plate was observed with any of the tester strains in either the presence or absence of S9 mix. All criteria for a valid study were met (Table 14; Table 13; Table 12; Table 11; Table 10; Table 9; Table 8; Table 7).

There were two values for TA98 strain with S9 mix, which fall below the acceptance range, but the difference was not significant. Hence, considered as normal values.

Positive Control showed > 2-fold increase in a number of revertant colonies in all the tester strains when compared to that of the negative control.

Carcinogenicity is due to damage and changes in DNA sequencing, gene mutation, chromosomal aberrations, and abnormalities. Natural antimutagenic agents lower the mutative action either by inactivating mutagens or by interfering with the process of mutagenesis (Maron & Ames, 1983). Bacterial reverse mutation assay was one of the renowned methods of determining the mutagenic potential of a target substance.

The results of our (OECD, 1997) Bacterial Reverse Mutation test with a method I (Plate incorporation) and method II (Preincubation) indicate that under the conditions of the study, the Polyherbal PF3 extract at a maximum dose of 5 mg/plate did not cause a positive increase in the mean number of revertant colonies per plate with any of the tester strains either in the presence or absence of metabolic activation (S9).

Hence, it can be concluded that Polyherbal extract at a maximum dose of 5 mg/plate showed no potential Genotoxic effect over any tester strains used in this study both in the presence (+S9) and absence of metabolic activation (-S9).

The research work was attributed to similar works carried (Resende, Vilegas, Santos, & Varanda, 2012) in determining the mutative potentialities of plant flavonoids.

Similar methods of evaluation of genotoxicity effects were incorporated in Agaricus blazei Murrill (Chang et al., 2012) and Antrodia cinnamomea (Wu et al., 2011), which were in support to the selected method of evaluation.

Conclusion

Annona Squamosa, Zingiber Officinalis, and Triticum Aestivum were found to be rich in flavonoids, and other phytoconstituents have shown antimutative properties may be by inactivating mutagens or by interfering in the process of mutagenisis. The optimum and in vivo model of antimutagenic studies of this polyherbal formulation need to be proven further to authenticate the effects.

[1] WHO 2018. World Health Organization. Fact sheet. Fact sheet

[2] Macdonald, JS . 1999. Toxicity of 5-fluorouracil. Oncology 13:33–44.

[3] Rexroth, G & Scotland, V . 1983. Cardiac toxicity of 5-fluorouracil. Medizinische Klinik 89(12):680–688.

[4] Avilés, A, Arévila, N, Díaz Maqueo, J C & Nambo, M J . 1993. Late Cardiac Toxicity of Doxorubicin, Epirubicin, and Mitoxantrone Therapy for Hodgkin’s Disease in Adults. Leukemia & Lymphoma 11(3-4):275–279.

[5] Manil, L, Mahieu, P & Couvreur, P . 1995. Acute Renal Toxicity of Doxorubicin (Adriamycin)-Loaded Cyanoacrylate Nanoparticles. Pharmaceutical Research 12(1):85–87.

[6] Gibaud, S, Andreux, J P, Weingarten, C, Renard, M & Couvreur, P . 1994. Increased bone marrow toxicity of doxorubicin bound to nanoparticles. European Journal of Cancer 30(6):90299–90301.

[7] Fraiser, L H, Kanekal, S & Kehrer, J P . 1991. Cyclophosphamide toxicity. Characterising and avoiding the problem. Drugs 42(5):781–795.

[8] OECD, . 1997. Organization for Economic Cooperation and Development. Bacterial Reverse Mutation Test. OECD Guidelines for testing of chemicals. 471.

[9] Maron, D M & Ames, B N . 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research/Environmental Mutagenesis and Related Subjects 113(3-4):90010–90019.

[10] Resende, F A, Vilegas, W, Santos, L C & Varanda, E A . 2012. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames) Test. Molecules 17(5):5255–5268.

[11] Chang, J B, Lu, H F, Liao, N C, Lee, C S, Yeh, M Y, Liu, C M & Chung, J G . 2012. Evaluation of genotoxicity and subclinical toxicity of Agaricus blazei murrill in the ames test and in histopathological and biochemical analysis. In Vivo 26:437–482.

[12] Wu, M F, Peng, F C, Chen, Y L, Lee, C S, Yang, Y Y, Yeh, M Y & Chung, J G . 2011. Evaluation of genotoxicity of Antrodia cinnamomea in the Ames test and the in vitro chromosomal aberration test. In Vivo 25:419–442.