Ethnobotanical Leaflets 12: 682-689. 2008.

 

 

Mutagenicity and Carcinogenicity Prediction of Compounds from Cardamom (Elettaria cardamom Maton.)

 

S.Balaji1 and B.Chempakam 2

 

Indian Institute of Spices Research, Calicut-673 012, Kerala, India

1Doctoral Research Associate, Bioinformatics centre

2Principal Scientist and Head - Biochemistry, Division of Crop Production

and Post Harvest Technology .

1Correspondance: e-mail: blast_balaji@rediffmail.com

 

Issued 12 September 2008

 

 

 

Abstract

            In silico approaches are currently not employed in any of the spices to study the toxicity. The aim of this study is to find the most efficacious molecule which does not have any adverse effects. In the present study one hundred and eight compounds from cardamom were used to predict mutagenicity and carcinogenecity. The results of these studies indicate that only four compounds are non-mutagenic and non-carcinogenic. The rest of the compounds do not have the characteristics necessary to become therapeutic agents have been identified early and prevented (i.e., the fail early, fail fast approach) from entering the drug development process.

 

Introduction

            Herbs and spices have been an essential factor in health care through the ages in all cultures. Many crude drugs are used medicinally because of their volatile oil content or other chemical constituents that posses biological activities. Cardamom is very popular as a spice and food additive because of its delicious flavour. The constituents of its volatile oil are responsible for the flavour and fragrance. It also possesses carminative, stomachic and antimicrobial actions. These biological activities bring about many advantages to the seasoned and prepared foods. Apart from this, cardamom finds application in the indigenous systems of medicine (Ravindran and Madhusoodanan, 2002).

            Cardamom seed oil is obtained naturally from dried ripe seeds of Elettaria cardamomum. The essential oil (2-8%) contains eucalyptol (cineol), sabinene, d,-terpineol and acetate, borneol, etc. The fixed oil (1-2%) consisits of glycerides of oleic, stearic, linolenic, palmitic, caprylic and caproic acids. It is used in the flavouring of liqueurs. Essential oil and their constituents (the resultant of secondary metabolism in plants) have been shown to be a potent source of botanical pesticide. The toxicity of a large number of essential oils and their constituents has been evaluated against a number of bruchid pests (Keita, et al., 2000, 2001, Tripathi et al., 2002). Plant essential oils and their constituents in relation to contact and fumigant insecticidal actions have been well demonstrated against stored product pests. Especially their main compounds monoterpenoids, offer promising alternatives to classical fumigants (Papachristos & Stamopoulos, 2003) and also have some effects on biological parameters such as growth rate, life span and reproduction (Pascual-Villalobos, 1996). Cardamom oil is shown to have antibacterial and antifungal action. Badei et al. (1991a,b) studied the chemical composition, physicochemical properties and anti-microbial activity of dried fruits of cardamom to assess the potential usefulness of cardamom oil as a food preservative. The antimicrobial effect of the oil was tested against 9 bacterial strains, 1 fungus and 1 yeast, the oil was 28.9 % as effective as phenol.

            To a small extent it is used in flavouring cigarette and tobacco (Ravindran and Madhusoodanan, 2002). Cardamom is used as an adjuvant to carminative drugs. It is officially recognized in British and US pharmacopoeias and used as an aromatic stimulant, carminative and flavouring agent. Cardamom seed oil is a common cosmetic ingredient. This material appears on the list of “Permitted Additives to Tobacco Products in the United Kingdom" (Department of Health, 2003) at a maximum level permitted for inclusion in cigarettes of 0.15 % w/w tobacco. However   cardamom seed oil (CAS No. 8000-66-6) is currently used worldwide at levels below 5 ppm in selected cigarette brands manufactured by Philip Morris International Inc., New York. Toxic effects on humans are not currently available, because as a food flavouring additive, the material has been assessed under the provisions of the Federal Food, Drug and Cosmetic Act, section 201 (s), by the Expert Committee of the USA Flavour and Extract manufacturer’s Association (FEMA), to be generally recognized as safe (GRAS) under current conditions of use. In contrast, it was found in literature that it can also be used to ease cigarette addiction. Eating a few seeds of cardamom can safely be recommended to initially minimize the number of cigarettes being smoked and slowly the smoker may give up the chronic addiction to chain smoking (Peter, 2001).

            Ironically, in a maximization test, a concentration of 4% of cardamom seed oil in petrolatum produced no sensitization reactions in 25 male volunteers (Kligman, 1973 cited in Opdyke, 1979). In a study of 25 workers in a spice factory, one worker was positive to cardamom on patch-test (Meding, 1993). In control test on 22 dermatitis patients without occupational exposure, one patient reacted to cardamom. No phototoxic effects were reported for cardamom seed oil (Urbach and Forbes, 1972 cited in Opdyke, 1979). But it has been identified the lethal dose in rat and rabbit as well as the genetic effects and mutations in bacteria. The present study was carried out to verify the contradictory statements pertaining to cardamom. It is well known that in silico approaches are comparatively cheaper than in vivo and in vitro screenings. Hence in the present study in silico methods were used to test one hundred and eight chemical structures from cardamom essential oil.

 

Methodology

 

Data collection

            The aim of this study is to screen the diverse array of chemical compounds for removing non-drug-like compounds from the drug discovery lifecycle in the early stages. “Fail early and fail fast” is the current paradigm that the pharmaceutical industry has adopted widely. To achieve this, 108 chemical compounds from cardamom were collected from literature of reported compounds and from the NCBI PubChem database (http://pubchem.ncbi.nlm.nih.gov/).

 

Ames mutagenicity assay

            Mortelmans and Zeiger (2000) described a short-term bacterial mutation assay caused by chemical substances. According to the data set of National Toxicology Program (NTP), the built biological model for toxicity prediction includes 3 strains: TA98, TA100, and TA1535.

 

Rodent carcinogenicity

            The Predictive Toxicology Challenge (PTC) was initiated for the development of advanced technology for predictive toxicology models. We have used computational models for carcinogenicity prediction created by Helma and Kramer (2003) with the data set of both National Toxicology Program (NTP) (Benigni, 1997) and Food and Drug Administration (FDA).

 

Results & Discussion

 

            The following compounds were found to be non-mutagenic in the biological model for 3 strains: TA98, TA100, and TA1535 [Table 1].

1-decanol, 1-heptanol, 1-hexanol, 1-octanol, a-ylangene, -guriunene, cedrol, citronellal, decanal, decyl acetate, dodecyl acetate, eicosanoic acid, farnesol, farnesyl acetone, geranyl acetone, humulene, octyl acetate, trans-2-cis-6-dodecadienal, trans-farnesol and undecan-2-one.

            The following compounds are found to be non-carcinogenic in the computational model for Rodent carcinogenicity prediction [Table 1].

1-heptanol, 1-hexanol, 2methyl-3-buten-2-ol, 2-methylbutanal, 3-methylbutanal, a,p-dimethylstyrene, a-terpinyl acetate, carvone oxide, decanal, delta-terpineol, ethyl 2-hydroxyhexanoate, hexanal, nonanal, oct-1-en-3-ol, octanal, p-dimethylstyrene, phenol, p-menth-8-en-2-ol, terpinyl acetate, tetrahydrolinalool, thymol, trans-2-butenal, trans-dec-2-enal, trans-nerolidol and trans-oct-2-enal.

            The mutagenecity and carcinogenicity prediction of the analyzed data set of 108 compounds yielded 1-heptanol, 1-hexanol and decanal, they were found to be non-mutagenic as well as non-carcinogenic. Hence, we conclude that these compounds may be a lead for drug discovery. All other compounds were predicted as mutagenic and carcinogenic.

            Toxicity of essential oil from cardamom was investigated against the cowpea weevil, Callosobruchus maculatus (Fab.) adults (an important pest of several pulses), through contact and fumigation bioassay (Mahfuz and Khalequzzaman, 2007). In the contact bioassay the toxicity of cardamom oil was higher than neem. In the fumigation bioassay, the efficacy in respect of the toxicity, cardamom oil was higher than neem and eucalyptus oils. This confirms that cardamom is a good fumigant.

            Currently, none of the human studies was conducted on the health effects of ingredients used in cigarette manufacture, studies have been conducted using scientifically accepted in vitro and in vivo toxicity assays with various ingredient mixtures. These studies show there is no meaningful difference in the composition or toxicity of smoke when the smoke from cigarettes with added ingredients is compared to the smoke from cigarettes without added ingredients. These findings are supported by similar studies from the published literature on burnt material (Gaworski et al., 1998, 1999; Carmines, 2002; Rustemeier et al., 2002 ; Roemer et al., 2002 ; Vanscheeuwijck et al., 2002). But in contrast, the in silico studies showed that most of the chemicals from the cardamom were mutagenic and carcinogenic [Table 1]. This is in concordance with the study of Meding (1993) and moreover several research works illustrated that essential oils and their constituents may have potential as alternative compounds to currently used fumigants (Huang et al., 2000; Tunc et al., 2000; Lee et al., 2001a, b).

 

Conclusion

            The results of these studies indicate that only four compounds are non-mutagenic and non-carcinogenic. The rest of the compounds do not have the characteristics necessary to become therapeutic agents have been identified early and prevented (i.e., the fail early, fail fast approach) from entering the drug development process. Because removing non-drug-like compounds from the drug discovery lifecycle in the early stages can lead to tremendous savings of resources (Cheng and Merz, 2003).

 

 

 

 

 

 

Table 1: Toxicity prediction profile of compounds from Cardamom.

 

Sl No

         Chemical Compounds

Ames Mutagenicity test

 

Rodent Carcinogenicity

 

 

 

-S9

+S9

Total

 

 

Total

 

 

TA98

TA100

TA1535

TA98

TA100

TA1535

Result

Mouse

Rat

Result

1

(E)-limonene oxide

+

-

+

-

+

+

+

+

+

+

2

(Z)-b-ocimene

+

-

-

+

-

-

+

+

+

+

3

1,3,8-menthatriene

+

-

-

+

+

-

+

+

+

+

4

1,4-cineole

-

-

-

-

-

+

+

+

+

+

5

1-decanol

-

-

-

-

-

-

-

+

-

+

6

1-heptanol

-

-

-

-

-

-

-

-

-

-

7

1-hexanol

-

-

-

-

-

-

-

-

-

-

8

1-nonanol

-

-

-

-

-

-

-

+

-

+

9

1-octanol

-

-

-

-

-

-

-

+

-

+

10

2,3-dehydro-1,8-cineole

-

-

-

-

-

+

+

+

+

+

11

2methyl-3-buten-2-ol

-

-

+

-

+

+

+

-

-

-

12

2-methylbutan-1-ol

-

+

+

-

-

+

+

+

-

+

13

2-methylbutanal

-

+

+

-

-

+

+

-

-

-

14

2-methylpropan-1-ol

-

+

+

-

-

+

+

+

-

+

15

3-methylbutanal

-

+

+

-

-

+

+

-

-

-

16

3-methylpentan-2-ol

-

-

+

-

-

+

+

+

+

+

17

4-thujanol

-

-

-

-

-

+

+

-

+

+

18

6-methyl-5-hepten-2-one

-

+

+

+

-

-

+

-

+

+

19

a-copaene

-

-

-

-

-

-

-

-

+

+

20

a,p-dimethylstyrene

+

-

-

+

+

-

+

-

-

-

21

a-ionone

-

-

-

-

-

+

+

+

-

+

22

a_phellandrene

+

-

-

+

+

-

+

+

+

+

23

a_pinene

-

-

-

-

+

-

+

-

+

+

24

a_terpinene

+

-

-

+

-

-

+

+

+

+

25

a_terpineol

-

-

-

-

-

+

+

-

-

-

26

a-terpinyl acetate

-

-

-

-

-

+

+

-

-

-

27

a-terpinyl propionate

-

-

-

-

-

+

+

+

-

+

28

a-thujene

-

-

+

-

+

-

+

-

+

+

 

 

TA98

TA100

TA1535

TA98

TA100

TA1535

Result

Mouse

Rat

Result

29

a-ylangene

-

-

-

-

-

-

-

-

+

+

30

b_caryophyllene

+

-

-

-

+

-

+

-

+

+

31

b-elemene

+

-

-

-

-

-

+

-

+

+

32

b-gurjunene

-

-

-

-

-

-

-

+

+

+

33

b_pinene

+

-

-

-

+

-

+

-

+

+

34

bornyl acetate

-

-

+

-

-

+

+

-

+

+

35

Camphene

+

-

-

-

+

-

+

-

+

+

36

Camphor

-

-

-

-

-

+

+

-

+

+

37

Carvacrol

+

-

+

+

+

+

+

-

-

-

38

Citronellol

-

-

-

-

-

-

-

+

-

+

39

carvone oxide

+

-

+

-

+

+

+

-

-

-

40

Cedrol

-

-

-

-

-

-

-

-

+

+

41

cis-carveol

-

-

-

+

-

+

+

+

-

+

42

cis-linalol oxide

-

-

+

-

+

+

+

+

+

+

43

cis-ocimene

+

-

-

+

-

-

+

+

+

+

44

Citronellal

-

-

-

-

-

-

-

+

-

+

45

Cubenol

-

-

-

-

-

+

+

+

-

+

46

Decanal

-

-

-

-

-

-

-

-

-

-

47

decyl acetate

-

-

-

-

-

-

-

+

-

+

48

Delta-cadinene

+

-

-

-

-

-

+

+

+

+

49

delta-terpineol

-

-

-

-

-

+

+

-

-

-

50

dodecyl acetate

-

-

-

-

-

-

-

+

-

+

51

eicosanoic acid

-

-

-

-

-

-

-

-

+

+

52

ethyl 2-hydroxyhexanoate

-

+

+

-

-

-

+

-

-

-

53

Farnesol

-

-

-

-

-

-

-

+

-

+

54

farnesyl acetone

-

-

-

-

-

-

-

+

+

+

55

gamma-cadinene

+

-

-

-

-

-

+

-

+

+

56

gamma-terpinene

-

-

-

+

-

-

+

+

+

+

57

geranyl acetone

-

-

-

-

-

-

-

-

+

+

58

germacrene-D

+

-

-

-

-

-

+

+

+

+

59

Globulol

-

-

-

-

+

+

+

-

+

+

60

g-murolene

+

-

-

-

-

-

+

-

+

+

 

 

TA98

TA100

TA1535

TA98

TA100

TA1535

Result

Mouse

Rat

Result

61

Guaiene

+

-

-

-

-

-

+

-

+

+

62

hexadecanoic acid

-

-

+

-

-

-

+

-

+

+

63

Hexanal

-

+

+

-

-

-

+

-

-

-

64

Humulene

-

-

-

-

-

-

-

+

+

+

65

isopiperitenol

+

-

-

+

-

+

+

+

-

+

66

Isosafrole

+

+

+

+

+

-

+

+

+

+

67

Limonene

+

-

-

+

-

-

+

-

+

+

68

Linalool

-

-

+

-

+

+

+

-

-

+

69

limonene-1,2-epoxide

+

-

+

-

+

+

+

+

+

+

70

linalyl acetate

-

-

+

-

-

+

+

+

+

+

71

menthatriene

+

-

-

+

+

-

+

+

+

+

72

methyl eugenol

-

-

+

+

+

+

+

+

+

+

73

Myrcene

-

-

+

+

-

-

+

-

+

+

74

Nerol

-

+

-

+

-

-

+

+

-

+

75

Nerolidol

-

-

-

-

-

-

-

-

-

-

76

neryl acetate

-

+

-

-

-

-

+

+

-

+

77

neryl propionate

-

+

-

-

-

-

+

+

-

+

78

Nonanal

-

-

+

-

-

-

+

-

-

-

79

oct-1-en-3-ol

-

+

+

-

-

-

+

-

-

-

80

octadecanoic acid

-

-

+

-

-

-

+

-

+

+

81

Octanal

-

-

+

-

-

-

+

-

-

-

82

octyl acetate

-

-

-

-

-

-

-

+

-

+

83

p-dimethylstyrene

+

-

-

+

+

-

+

-

-

-

84

Perillene

-

+

+

+

-

+

+

+

+

+

85

Phenol

+

+

+

+

+

-

+

-

-

-

86

Piperitenone

+

+

-

+

-

+

+

+

+

+

87

p-menth-8-en-2-ol

-

-

-

-

-

+

+

-

-

-

88

Sabinene

+

-

+

+

+

-

+

+

+

+

89

terpinen-4-ol

+

-

-

-

-

+

+

+

-

+

90

Terpinolene

-

-

-

+

-

-

+

+

+

+

91

terpinyl acetate

-

-

-

-

-

+

+

-

-

-

92

tetrahydrolinalool

-

-

+

-

+

+

+

-

-

-

93

Thymol

+

+

-

+

+

+

+

-

-

-

94

T-muurolol

-

-

-

-

-

+

+

+

+

+

95

Toluene

 

 

 

 

 

 

 

 

 

 

96

trans-2-butenal

+

+

+

+

-

+

+

-

-

-

97

trans-2-cis-6-dodecadienal

-

-

-

-

-

-

-

+

+

+

98

trans-carveol

-

-

-

+

-

+

+

+

-

+

99

trans-dec-2-enal

-

+

-

-

-

-

+

-

-

-

100

trans-farnesol

-

-

-

-

-

-

-

+

-

+

101

trans-linalool oxide

+

-

+

-

-

+

+

+

+

+

102

trans-nerolidol

-

-

-

-

-

+

+

-

-

-

103

trans-ocimene

+

-

-

+

-

-

+

+

+

+

104

trans-oct-2-enal

-

+

+

+

-

-

+

-

-

-

105

trans-p-mentha-2,8-dien-1-ol

-

+

-

+

-

-

+

+

+

+

106

Tricyclene

-

-

-

-

+

-

+

-

+

+

107

undecan-2-one

-

-

-

-

-

-

-

+

-

+

108

Valencene

+

-

-

-

-

-

+

-

+

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Note: ’+’ indicates the presence, ‘-’ indicates the absence of mutagenicity and carcinogenicity.

 

 

 

References:

 

Badei, A.Z.M., El-Akel, A.T.M. and Morsi, H.H.H. Evaluation of chemical, physical and antimicrobial properties of cardamom essential oil. Bull. Faculty of Agri., University of Cairo, 1991, 42(1), 183-197.

 

Badei, A.Z.M., Morsi, H.H.H. and El-Akel, A.T.M. Chemical composition and antioxidant properties of cardamom essential oil. Bull. Faculty of Agri., University of Cairo, 1991, 42(1), 199-215.

 

Benigni, R. The first US National Toxicology Program exercise on the prediction of rodent carcinogenicity: definitive results. Mutation Res., 1997, 387, 35-45.

 

Carmines, E.L. “Evaluation of the Potential Effects of Ingredients Added to Cigarettes. Part I: Cigarette Design, Testing Approach and Review of Results” Food and Chemical Toxicology, 2002, 40:77-91.

 

Cheng, A. and Merz, K.M Jr. Prediction of aqueous solubility of a diverse set of compounds using quantitative structure-property relationships. J Med Chem., 2003, 46, 3572-80.    

 

Gaworski C. L., Heck J. D., Bennett M. B., Wenk M. L. Toxicologic evaluation of flavor ingredients added to cigarette tobacco: skin painting bioassay of cigarette smoke condensate in SENCAR mice. Toxicology, 1999,139 1-17.

 

Gaworski CL, Dozier MM, Heck JD, et al. Toxicologic evaluation of flavor ingredients added to cigarette tobacco---13-week inhalation exposures in rats. Inhalation Toxicol, 1998, 10:357-381.

 

Helma, C. and Kramer, S. A survey of the Predictive Toxicology Challenge 2000-2001. Bioinformatics., 2003, 19,1179-1182.

 

Huang, Y., Lam, S. L. & Ho, S. H. Bio-activities of essential oil from Elletaria cardamomum (L.) Maton. to Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst). J. stored Prod. Res. 2000, 36 (2): 107-117.

 

Keita, S. M., Vincent, C., Belanger, A. & Schmit, J. P. Effect of various essential oils on Callosobruchus maculatus (F.) [Coleoptera: Bruchidae]. J. stored Prod.Res. 2000, 36: 355–364.

 

Keita, S. M., Vincent, C., Schmit, J. P. Arnason, J. T. & Bélanger, A. Efficacy of essential oil of Ocimum basilicum L. and O. gratissimum L. applied as an insecticidal fumigant and powder to control Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). J. stored Prod. Res. 2001, 37: 339-349.

 

Kligman, A.M. Report to RIFM, 13 June. (Cited in Opdyke, 1979) 1973.

 

Lee, B.H., Choi, W.S., Lee, S. E. & Park, B. S. Fumigant toxicity of essential oils and their constituent compounds towards the rice weevil, Sitophilus oryzae (L). Crop Protec. 2001, 20: 317–320.

 

Lee, S. E., Lee, B. H., Choi, W. S., Park, B. S., Kim, J. G. & Campbell, B. C. Fumigant toxicityof volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L.). Pest Manag. Sci. 2001, 57: 548–553.

 

Mahfuz, I and Khalequzzaman, M. Contact and Fumigant Toxicity of Essential Oils Against Callosobruchus maculatus. Univ.j.zool. Rajshahi Univ. 2007, 26:63-66.

 

Meding, B. Skin symptoms among workers in a spice factory. Contact Dermatitis, 1993, 29:202-205.

 

Mortelmans, K and Zeiger, E. The Ames Salmonella/microsome mutagenicity assay, Mutation Res., 2000, 455, 29-60.

 

Noleau, I. and Toulemonde, B. Volatile constituents of cardamom (Elettaria cardamomum Maton) cultivated in Costa Rica. Flavour Fragr. J., 1987, 2, 123-127.

 

Opdyke, D.L.J. Monographs on Fragrance Raw Materials. Cardamom Oil, pp. 1979, 180-181.

 

Papachristos, D.P. & Stamopoulos, D. C.. Selection of Acanthoscelides obtectus (Say) for resistance to lavender essential oil vapour. J. stored Prod. Res. 2003, 39: 433-441.

 

Pascual-Villalobos, J. M. Evaluation of the insecticidal activity of Chrysanthemum coronarium L. plant extracts. Bol. Sanidad Veg. Plagas. 1996, 22: 411–420.

 

Peter K.V. (Ed).    Handbook of herbs and spices, Woodhead publishing in Food Science and Technology, Woodhead Publishing Limited, Abington, Cambridge, England. 2001.

 

Ravindran P.N, Madhusoodanan, K.J. (Eds). Cardamom: The Genus Elettaria. Medicinal and Aromatic Plants – Industrial Profile. London, United Kingdom: Taylor and Francis. 2002.

 

Roemer, E., Tewes, F.J., Meisgen, T.J., Veltel, D. and Carmines, E.L.  “Evaluation of the Potential Effects of Flavor Ingredients Added to Cigarettes. Part 3. In Vitro Genotoxicity and Cytotoxicity” Food and Chemical Toxicology, 2002, 40:105-111.

Rustemeier K, Stabbert R, Haussmann HJ, Roemer E, Carmines EL. “Evaluation of the Potential Effects of Ingredients Added to Cigarettes Part II. Chemical Smoke Composition” Food and Chemical Toxicology, 2002, 40:93 - 104.

 

Tripathi, A. K., Prajapati, V., Verma, N., Bhal, J. R., Bansal, R. P., Khanuja, S.P.S. & Kumar, S. Bioactivities of the leaf essential oil of Curcuma longa (Var. Ch-66) on three species of stored-product beetles (Coleoptera). J. Econ. Entomol. 2002, 95 (1): 183-189.

 

Tunc, I., Berger, B. M., Erler, F. & Dagli, F. Ovicidal activity of essential oils from five plants against two stored-product insects. J. stored Prod. Res. 2000, 36: 161-168.

 

Urbach, F. and Forbes, P.D. Report to RIFM, 8 February. (Cited in Opdyke, 1979). 1973.

 

Vanscheeuwijck P.M., Teredesai A., Terpstra P.M., Verbeeck J., Kuhl P., Gerstenberg B., Gebel S., Carmines E.L. Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity. Food Chem Toxicol. 2002, 40(1):113-31.