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Inhibitory effects of Nigella sativa and saffron (crocus sativus) on chemical carcinogenesis in mice a

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M.J. Salomi , Satish C. Nair & K.R. Panikkar

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Amala Cancer Research Centre , Amala Nagar, Trichur, Kerala, 680 553, India Published online: 04 Aug 2009.

To cite this article: M.J. Salomi , Satish C. Nair & K.R. Panikkar (1991) Inhibitory effects of Nigella sativa and saffron (crocus sativus) on chemical carcinogenesis in mice, Nutrition and Cancer, 16:1, 67-72, DOI: 10.1080/01635589109514142 To link to this article: http://dx.doi.org/10.1080/01635589109514142

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Inhibitory Effects of Nigella sativa and Saffron (Crocus sativus) on Chemical Carcinogenesis in Mice Downloaded by [New York University] at 11:39 25 May 2015

M.J. Salomi, Satish C. Nair, and K.R. Panikkar

Abstract Topical application of Nigella sativa and Crocus sativus extracts (common food spices) inhibited two-stage initiation/promotion [dimethylbenz[a]anthracene (DMBA)/croton oil] skin carcinogenesis in mice. A dose of 100 mg/kg body wt of these extracts delayed the onset of papilloma formation and reduced the mean number of papillomas per mouse, respectively. The possibility that these extracts could inhibit the action of 20-methylcholanthrene (MCA)induced soft tissue sarcomas was evaluated by studying the effect of these extracts on MCA-induced soft tissue sarcomas in albino mice. Intraperitoneal administration of Nigella sativa (100 mg/kg body wt) and oral administration of Crocus sativus (100 mg/kg body wt) 30 days after subcutaneous administration of MCA (745 nmol × 2 days) restricted tumor incidence to 33.3% and 10%, respectively, compared with 100% in MCA-treated controls. (Nutr Cancer 16, 67-72, 1991)

Introduction

Chemical carcinogenesis studies have gained a lot of interest because of their importance in understanding the induction of cancer (1). The initiation/promotion technique of chemical carcinogenesis using a promoter results in the formation of multiple papillomas after a short latency period (2). In the two-stage carcinogenesis initiation, there is an irreversible step in which change occurs in the genes controlling epidermal differentiation (3). On the other hand, promotion causes an alteration in the epigenetic mechanism (4). Curcumin, the coloring matter from turmeric, a food spice (5), and carotene from carrots (6) have been shown to inhibit tumor promotion in mice. Nigella sativa seeds and saffron (Crocussativus), common food spices, are used to treat various diseases by the Indian system of medicine (7).

The authors are affiliated with the Amala Cancer Research Centre, Amala Nagar, Trichur 680 553, Kerala, India.

Copyright © 1991, Lawrence Erlbaum Associates, Inc.

Inhibitors of chemical carcinogens and antimutagens can have a profound effect on the expression of experimental tumors (8). For this paper, we investigated the effect of Nigella sativa and saffron extracts on the inhibition of chemically induced tumors in mice. Materials and Methods

Chemicals

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7,12-Dimethylbenz[a]anthracene (DMBA) was obtained from Sigma Chemical (St. Louis, MO) and 20-methylcholanthrene (MCA) from ICN Pharmaceuticals (New York). Croton oil was obtained from the seeds of Croton tiglium by light petroleum extraction. The natural products Nigella sativa seeds (purchased from a local market) and saffron (Crocus sativus) (Government Emporium, Kashmir, India) were stored at 4°C. These were extracted and purified as described next. Extraction and Purification of Natural Products Nigella sativa seeds were washed in distilled water, dried in an oven at 60°C, and powdered. Five grams of powder were extracted with 100 ml of 95% methanol, by keeping overnight. The extract was filtered, and the filtrate was concentrated to remove methanol completely. This crude extract was passed through a silica gel column (35 cm x 20 cm) and eluted with methanol-water (9:1) (250 ml). The active fractions were pooled together, concentrated, and separated on Whatman no. 1 filter paper (ascending paper chromatography), using chloroform as the solvent. The active fraction present near the solvent front (having an Rf value of 0.9) was eluted with methanol, concentrated, and used for further studies. One gram of saffron was extracted with 50 ml of 95% ethanol (3 times) overnight at 28 °C with continuous stirring. The pooled extracts were evaporated under reduced pressure to a known volume (5 ml) and loaded on a silica gel (Glaxo, India) column as above and eluted successively with petroleum benzene (250 ml), chloroform (250 ml), methanol (250 ml), and water (250 ml). The water fraction (yellow color) containing the required compound was further separated by ascending paper chromatographic technique with butanol-acetic acid-water (6:1:2 vol/vol/vol) as the solvent system. The active spot (faint yellow color) nearer the base (Rf = 0.2) was cut, eluted with methanol (95%) overnight, evaporated to free alcohol, dried to a powder form, and stored at 4°C. All reagents used were of analytic grade. Cytotoxicity Assay In Vitro The activity of the active fractions were ascertained by the cytotoxicity assay at each stage of purification as described by us earlier (9). Briefly, the finally concentrated (alcohol-free) powder was weighed and redissolved in ethanol (0.5 ml) and then reconstituted in phosphate-buffered saline (pH 7.4) to a concentration of 1 mg/ml. The concentration of alcohol was between 0.05% and 1.0% and did not interfere with the assay procedure. Daltons lymphoma ascites (DLA) tumor cells, previously grown in the peritoneal cavity of albino mice, were asceptically aspirated and washed with normal saline. DLA cells (1 X 106) were incubated with various concentrations of the reconstituted sample (Crocus sativus and Nigella sativa) for three hours at 37°C. The percentage of dead cells was determined by the trypan blue exclusion method, and the minimum concentration required to produce 50% cytotoxicity was ascertained. Fifty percent cytotoxicity was obtained at a concentration of 7 and 0.5 jtg of the active fractions from Crocus sativus and Nigella sativa, respectively.

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Animals Inbred strains of male Swiss albino mice (9-10 wks, 18-20 g) obtained from Amala Cancer Research Centre Animal Colony (Kerala, India) were used for the experiments. Groups of 10 mice in each set were used for each experiment. Skin Carcinogenesis Studies Dorsal skin areas between the cervical and caudal portions (2.5 X 4 cm) of mice were shaved with electric shears one to two days before the experiment (5). Animals showing no regrowth of hair were used.

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Tumor Induction and Treatment A single dose of DMBA [450 nmol in 0.1 ml dimethylsulfoxide (DMSO)] was topically applied as an initiator. Two weeks later, croton oil (50 /*1) was applied topically on the same site, twice weekly for six weeks. The application of croton oil was preceded by the application of Nigella sativa or saffron extract at a dose of 100 mg/kg body wt (2 mg in 0.1 ml DMSO) on the back of treated groups. The controls were treated with 0.1 ml DMSO for the same period. Soft Tissue Sarcoma Studies All the mice were subcutaneously injected with a dose of 745 nmol of MCA in 0.1 ml DMSO consecutively for two days. After 30 days of tumor initiation using MCA, the treated groups received Nigella sativa intraperitoneally or saffron extract orally using a gavage, at a dose of 100 mg/kg body wt (2 mg in 0.1 ml sterile physiological saline). The controls were treated with sterile saline (0.1 ml). Soft tissue sarcomas appeared in eight weeks. Tumor diameter was calculated by using the following formula (see Ref. 10). _ ,. . length (cm) + width (cm) i Tumor diameter (cm)x = — 2 — - — — —Results

The results of the effect of Nigella sativa and saffron extracts on skin carcinogenesis induced by DMBA is given in Table 1. Over half (56.6%) of the animals subjected to the initiation/promotion technique using DMBA and croton oil developed visible papillomas in a span of six weeks. On the 12th week of observation, 90% of the untreated control mice developed papillomas, compared with 76.6% and 50% for the groups treated with Nigella sativa and saffron extract, respectively. A significant decrease in the mean number of papillomas per mouse was observed in the treated groups: 1.3 papillomas/mouse in the group treated with Nigella sativa and 1.0 papillomas/mouse in the group treated with saffron. The number of papillomas varied between two and five per mouse (mean 2.4/mouse) in the untreated control group. These extracts further showed significant inhibitory effect on the growth of soft tissue sarcomas induced by MCA (Table 2). Palpable tumors appeared in 26.6% of mice by the eighth week in the untreated (MCA) controls posttumor initiation. The tumor incidence in carcinogenic controls (MCA) reached 100% and 33.3% and 10% in the groups treated with Nigella sativa and saffron after 12 weeks. None of the control animals survived up to 20 weeks. The mice treated with the spices developed tumors in 20 weeks (43.3% for Crocus sativus and 56.6% for Nigella sativa). Another point of interest was that after the first appearance of tumors in the control group, these animals could survive only up to a maximum of 24 days, whereas the treated mice survived up to 47 days.

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Table 1. Effect of Crocus sativus and Nigella sativa Extracts on DMBA-Induced Papillomas in Mice In Vivo" Percentage of Mice With Papillomas Treatment Initiator

Promoter

DMBA (450 nmol in 0.1 ml DMSO)

Croton oil

DMBA (450 nmol in 0.1 ml DMSO)

Croton oil

DMBA (450 nmol in 0.1 ml DMSO)

Croton oil

Drugs

Crocus sativus extract (100 mg/ kg body wt) Nigella sativa extract (100 mg/ kg body wt)

Mean Number of Papillomas/Mouse*

6th Week

9th Week

12th Week

6th Week

9th Week

12th Week

56.6

76.6

90

0.80 ± 0.5

2.0 ± 0.6

2.4 ± 0.5

26.6

26.6

50

0.26 ± 0.13

0.57 ± 0.27

1.0 ± 0.3c

33.3

33.3

76.6

0.53 ± 0.35

0.53 ± 0.26

1.3 ± 0.6"

a: DMBA-induced papillomas were initiated by topical application of DMBA and were promoted with croton oil 2 wks after initiation, biweekly for 6 wks. Crocus sativus or Nigella sativa extracts were applied prior to croton oil application for 6 wks. Mice treated twice weekly with DMSO, Crocus sativus, or Nigella sativa extract without DMBA did not develop tumors. b: Values are means ± SD from 3 separate experiments using 10 male albino mice/group/expt. c: Significance was p < 0.02. d: Significance was p < 0.1.

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Table 2. Effect of Crocus sativus and Nigella sativa Extracts on 20-Methylcholanthrene-Induced Soft Tissue Sarcomas in Mice In Vivo" Mean Tumor Diameter,*

Percentage of Mice With Tumors 8th

Treatment MCA (745 nmol sc x 2 days) MCA (745 nmol sc x 2 days) MCA (745 nmol sc x 2 days)

Drugs Sterile saline (0.1 mlip/oral) Crocus sativus extract (100 mg/ kg body wt oral x 5 days) Nigella sativa extract (100 mg/ kg body wt ip x 5 days)

Week

12th Week

cM

20th Week

8th Week

12th Week

0.83 ± 0.41

3.15 ± 0.52

26.6

100

0

10

43.3

0.93 ± 0.26c

0

33.3

56.6

1.47 ± 0.48d

a: Sarcomas were nitiated with 20-methylcholanthrene (MCA). Crocus sativus (saffron) and Nigella sativa extracts were administered at dose of 100 mg/kg body wt (2 mg in 0.1 ml saline) 30 days after tumor initiation. Mice treatec with DMSO, Crocus sativus, or Nigella sativa extract in absence of MCA did not develop tumors. b: Values are means ± SD from 3 experiments using 10 male albino mice/group/expt. c: Significance was p < 0.005. d: Significance was p < 0.02.

Discussion

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Saffron was found to contain water-soluble carotenoids (11). Many plant products, especially phenols, cinnamic acid derivatives, etc., have shown an inhibitory effect on tumor-promoting activity, possibly due to the oxygen-scavenging properties associated with these compounds (5). Our studies showed that the application of Nigella sativa and saffron extract reduced the expression of papillomas and also delayed the onset of papilloma formation in mice induced by DMBA followed by croton oil promotion. An interesting observation was that 90% of the control animals subjected to initiation promotion protocol developed papillomas. Oral administration of saffron extract and intraperitoneal administration of Nigella sativa extract could effectively prevent the incidence of soft tissue sarcomas and reduce the tumor diameters in treated groups, initiated by MCA. The previous studies indicate the effect of these natural products in inhibiting chemically induced skin carcinogenesis. This study is of importance because of the widespread uptake of spices and other plant products by the human population. Acknowledgments and Notes The authors thank the Council of Scientific and Industrial Research (CSIR) (New Delhi, India) for granting a Senior Fellowship to M.J. Salomi and to the Indian Council of Medical Research (ICMR) (New Delhi) for giving financial assistance in this work. Submitted 25 August 1990; accepted in final form 31 December 1990.

References 1. Slaga, TJ: "Overview of Tumor Promotion in Animals." Environ Health Perspect 50, 3-14, 1983. 2. Slaga, TJ: "Sencar Mouse Skin Tumorigenesis Model Versus Other Strains and Stocks of Mice." Environ Health Perspect 68, 27-32, 1986. 3. Dalmain, A, Ramsden, M, Bowgen, GT, and Smith, J: "Activation of the Mouse Cellular Harvey-ras Gene in Chemically Induced Benign Skin Papillomas." Nature 307, 658-660, 1984. 4. Hennings, H, Shores, R, Wenk, ML, Spangler, EF, Tarone, ML, et al.: "Malignant Conversion of Mouse Skin Tumours Is Increased by Tumour Initiations and Unaffected by Tumour Promotors." Nature 304, 67-69, 1983. 5. Tuan Huang, M, Smart, RC, Wong, CQ, and Conney, AH: "Inhibitory Effect of Curcumin, Chlorogenic Acid, Caffeic Acid, and Ferulic Acid on Tumour Promotion in Mouse Skin by TPA." Cancer Res 48, 5941-5946, 1988. 6. Foote, CS: "Carotenoids." In Pathology of Oxygen, A Autor (ed). New York: Academic, 1982, pp 21-24. 7. Nadkarni, KM: "Crocus sativus, Nigella sativa." In Indian Materia Medica, KM Nadkarni (ed). Bombay, India: Popular Prakashan, 1976, pp 386-411. 8. Wattenberg, LW: "Chemoprevention of Cancer." Cancer Res 45, 1-8, 1985. 9. Nair, SC, and Panikkar, KR: "Antitumour Principles From Ixora javanica." Cancer Lett 49, 121-126, 1990. 10. Abdel-Galil, AM: "Preventive Effect of Vitamin C (L-Ascorbic Acid) on Methylcholanthrene-Induced Soft Tissue Sarcoma in Mice." Oncology 43, 335-337, 1986. 11. Pfander, H , and Witwer, F: "Untersuchungen Zur Carotinoid—Zusammensetzung in Safran." Helv Chim Acta 581, 1608-1620, 1975.

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Inhibitory effects of Nigella sativa and saffron (Crocus sativus) on chemical carcinogenesis in mice.

Topical application of Nigella sativa and Crocus sativus extracts (common food spices) inhibited two-stage initiation/promotion [dimethylbenz[a]anthra...
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