effects of saffron on vitamin a levels and its antitumour activity on the growth of solid tumours in...
TRANSCRIPT
Int. J. Pharmacog., 32 (1994). No. 2, pp. 105-114 0925- 16 18/94/3202-0105$6.00 0 Swets & Zeitlinger
Effects of Saffron on Vitamin A Levels and its Antitumour Activity on the Growth of Solid Tumours in Mice
S.C. NAIR*, C.D. VARGHESE', K.R. PANIKER', S.K. KURUMBOOR and R.K. PARATHOD
University of Minnesota Medical School, 3-249, Millard Hall, 435, Delaware Street SE., Minneapolis, Minnesota 55455, U.S.A.
'Amala Cancer Hospital and Research Centre, Amalanagar (P.0) 680553, Trichur, Kerala, India
ABSTRACT
Inhibitory effects of saffron extract, a commonly used food spice, were studied against subcutane- ously inoculated solid Dalton's lymphoma ascites (DLA), Ehrlich ascites carcinoma (EAC) and Sarcoma-I80 (S-180) tumours in mice. Oral administration of 100 m g k g of the saffron extract significantly inhibited the growth of DLA and S-180 solid tumour by 87 and 41%. respectively. A dose of 150 mglkg delayed the onset of tumour formation and inhibited further growth of a delayed Dalton's lymphoma ascites solid tumour by 80%. Chromium release cytotoxicity assay reflected the sensitivity of DLA and S-180 tumour cells to cytolysis (in vitro) in the presence of the extract. Estimation of serum vitamin A and p-carotene levels from S-180 tumour bearing mice treated with saffron extract indicated elevated serum levels of serum vitamin A and p-carotene. Chemical analy- sis of the antitumour compound from saffron extract indicated that crocin (a natural carotenoid) was responsible for the observed biological effects. Crocin probably induces its therapeutic antitumour effect by its provitamin A activity and/or its antioxidant activity or by modulating the functional levels of other antioxidants.
INTRODUCTION
Saffron (Crocus sativus L., Iridaceae) is a commonly used food spice in Spain, Germany, India and other parts of the world. Foods are known to contain possi- ble cancer preventive factors (Ames et al., 1987). The possible anticarcinogenic activity of various dietary factors and micronutrients has been extensively stud- ied in relation to cancer (Henderson et al., 1991). A significant reduction in the incidence of lung and other epithelial cancers have been reported with increasing consumption of p-carotene, present in common fruits and vegetables (Henderson et al., 1991). It has been reported that patients with lung cancers or smoking related cancers have lower levels of serum p-carotene than healthy controls
'Address for correspondence: Dr. S.C. Nair, 600 S, 42nd Street, Omaha, NE 68198-6805, U.S.A.
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(Helzlsouer et al., 1989). Curcumin, the colouring matter from turmeric, a com- mon food spice, inhibited the expression of chemically-induced skin tumours (Huang et al., 1988) and forestomach tumours in mice (Nagabushan and Bhide, 1987). The Indian system of medicine uses the extracts of spices such as saffron, black cumin and ginger to treat various diseases including cancer (Nadkarni, 1976; Panikkar et al., 1986). Some of the compounds isolated from natural products were found to be helpful in preventing carcinogen activation and subse- quently inhibition in the expression of tumours (Wattenberg, 1985). We have reported earlier that saffron extract inhibited the growth of chemically-induced papillomas and soft tissue fibrosarcomas in mice (Salomi et al., 1991a). Oral administration of saffron extract also prevented the growth of transplanted as- cites tumours in mice (Nair et al., 1991a).
In this paper we have investigated the antitumour activity of saffron extract against experimentally-induced subcutaneous solid tumours in mice and the role of p-carotene in mediating the antitumour effect.
MATERIALS AND METHODS
Tumour cells Daltons lymphoma ascites (DLA), Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) tu- mour cells were a gift from the National Cancer Centre, Calcutta and Cancer Research Institute, Bombay, respectively, and were propagated in male albino mice by i.p weekly passages. Tumour cells were aspirated from respective turnour bearing mice, washed in sterile saline and counted under sterile conditions as described earlier (Nair and Panikkar, 1990).
Animals Inbred strains of male Swiss albino mice (9-10 weeks, 18-22 g) obtained from our animal facility were used for the experiment. The mice were kept in ventilated cages and maintained on standard mouse feed (Lipton, India).
Radioisotope slCr (Sodium chromate) was obtained from BARC, Bombay, and kept at - 70" C.
Drugs and chemicals Saffron (Crocus sativus) was purchased from the Government Emporium, Kashmir, India. Saffron was extracted and purified by the method described earlier (Salomi et al., 1991a). All other reagents used in this study were of the analytical grade.
Chromium release cytotoxicity assay Chromium release cytotoxicity assay (Nair et al., 1 9 9 1 ~ ) was performed using DLA, EAC and S-180 tumour cells. Tumour cells (1.5 X lo7) were incubated with 600 FCi of chromium in 1 ml MEM for 1 hour in siliconised glass tubes at 37°C. After the incubation, the tubes were centrifuged at 1000 rpm for 5 min and washed twice with warm MEM (Eagle's minimum essential medium) (37°C ), The supernatant was discarded and checked for radioactivity. The cell pellets were diluted to 1x106 cells/ ml in a total of lml, containing 10% goat serum. Drug in various concentrations was added to each of the tubes. The tubes for control (without drug) and total (chromium) release contained MEM or 1N
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SAFFRON EFFECTS ON VITAMIN A AND ANTITUMOUR ACTIVITY 107
HCI, respectively. All the tubes were then incubated for three hours at 37°C. After incubation, Iml MEM was added to each of the tubes, and they were centrifuged at 1200 rpm for 10 min. One ml of the supernatant was withdrawn from each tube and counted in a gamma counter (Electronic Corpora- tion, India). The % of cytolysis (T) was calculated using the formula (Brunner et al., 1976):
Mean experimental release (cpm)-Mean spontaneous release (cpm)
Mean total release (cpm)-Mean spontaneous release (cpm) % T = x 100
Solid tumour experiments Daltons lymphoma ascites (DLA) solid tumour DLA tumour cells (lo6) were subcutaneously injected on the right hind leg of mice and randomized into four groups. Each experiment consisted of six micelgroup. Group 1 received (100 pl) sterile saline orally , Groups 2 and 3 received 100 and 50 mg/kg, respectively, of saffron extract, for nine days successively, orally, once a day using a gavage one day after the transplantation of tumour cells. Group 4 was treated with 150 mg/kg of saffron extract, orally as above, but nine days after the tumour inoculum (Nair and Panikkar, 1990; Nair et al., 1992).
Ehrlich ascites carcinoma (EAC) solid tumour EAC solid tumours were induced in mice by the same procedure as above by injecting lo6 tumour cells. Two groups of mice consisting of six mice/group were used for the experiment. Group 5 was the untreated control tumour-bearing mice which received (100 pl) sterile saline. Group 6 was treated with 100 mg/kg of saffron extract orally for nine days as stated above.
Sarcoma-180 (S-180) solid tumour S-180 solid tumours were also induced by employing the above protocol. Mice were divided into three groups, group 7 was the saline (100 pl) treated tumour bearing control and groups 8 and 9 were treated with 100 and 50 mgkg, respectively, of saffron extract, daily for nine days, as described above.
Assessment of tumour volume (Vt) The antitumour activity of the drug was assessed by comparing the tumour volume between the control and treated groups using the formula (Nair and Panikkar, 1990) at an interval of five days for thirty days.
Vt = 4/3 x (R1)2 x (R2) [where RI and R2 are the major and minor radius at right angles to each other]
Estimation of Beta-carotene and vitamin A levels in mice Beta-carotene levels in treated mice were estimated in the form of serum vitamin A by the method of Neeld and Pearson (1963) using trifluoroacetic acid. Four groups of mice [A, B, C and D (20 mice per group)] were used for the experiment. Sarcoma-180 solid turnours were induced in these groups of mice as mentioned above. Groups A and B were the normal and tumour controls which received sterile saline during the same period. Group C and D received 50 and 100 mglkg of saffron extract in (100 pl) sterile saline orally for nine days successively using gavage. The blood was collected by sacrificing ten mice from each group on day 10 and 30, respectively. Heparinized blood was collect- ed by heart puncture from each of the groups, pooled and frozen at -70°C until ready for analysis. Two ml of serum was transferred to stoppered glass test tubes (Borosil, Bombay) and immediately followed by the addition of 2 ml of 95% ethanol and 3 ml of petroleum ether (30 - 5OOC) and mixed on a vortex mixer to ensure complete extraction of p-carotene and vitamin A. The tubes were then centrifuged at 2100 rpm for three minutes and 2 ml of the petroleum ether layer was pipetted and p- carotene read at 450 nm. The petroleum ether layer was then evaporated on a waterbath at 40-45OC. The residue was dissolved in 100 pl of chloroform and 100 pl of acetic anhydride and immediately
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read at 620 nm after adding 1 ml of trifluoroacetic acid (TFA) reagent. The blank was made up of 1 ml of chloroform and the same amount of TFA. Beta-carotene levels and vitamin A were estimated by comparing the UV absorption of the samples with that for standard P-carotene and vitamin A and the values expressed in the form of kg/IOO ml serum of p-carotene and vitamin A, respectively.
RESULTS
Chromium release cytotoxicity assay This assay was conducted to ascertain the extent of cytolysis after cellular mem- brane damage and the release of previously internalised chromium from tumour cells. At a saffron extract concentration of 15 pg/ml, 28.54+4.6, 14.8k0.76 and 33.7k3.35 percent specific cytolysis of DLA, EAC and S-180, respectively, were observed (Table 1). Maximum specific cytolysis of the tumour cells was ob- served at 30 pg/ml (DLA= 60+5.39%, EAC=24.5k4.3% and S180=65.2+6.28%). The total count was 16642 cpm, the mean total release (1N HCI) was 7499, and mean spontaneous release was 1897 cpm, from three experiments in duplicate.
Inhibitory effects on solid tumours DLA and S- 180 solid tumours responded well to the treatment using saffron extract as compared to EAC solid tumour. Saffron extract, at an oral dose of 100
Table 1. Chromium release cytotoxicity assay to ascertain the cytolytic potential of saffron extract to tumour cells in v i m .
Tumour Extract Mean CPM f S D Mean Percentage Mean Specific Cells Concentration Cytolysisf SD Cytolysis
( M m U
DLA 0 (Control) 2001f 49 1.86rt0.88 _ _ 7.5 295 lf185 18.8f3.3 16.94f3.2 15 3604f308 30.425.5 28.54f4.6 30 5364f351 61.8f6.2 60.015.39
_ _ EAC 0 (Control) 202M110 2.2f1.96 7.5 2232f 88 5.9f 1.59 3.7f2.6 15 2779f 68 17.0f2.5 14.8f0.76 30 3397f234 26.7k4.19 24.5f4.3
S- 180 0 (Control) 2013f 23 2.06rt0.41 _ _ 15.1k3.45 13.06f3.8 7.5 2745f192
15 3906f1 8 1 35.8rt3.2 33.7f3.35 30 5559f366 65.3k6.55 65.2f6.28 .
Values are MeanfSD from three separate experiments in duplicate. *P<O.OOl as compared with controls.
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SAFFRON EFFECTS ON VITAMIN A AND ANTITUMOUR ACTIVITY 109
mg/kg, significantly (P<O.OOl) inhibited the growth of subcutaneously trans- planted DLA solid tumour and delayed the onset of tumour formation while 50 mg/kg was less effective (P< 0.01). Administration of 150 mg/kg of saffron extract inhibited the growth of a ten day old tumour (P<O.OOl) significantly (Figure 1). The percentage inhibition in tumour growth for the treated groups which received 100 mg/kg and 150 mg/kg of saffron extract were 87% and 80% as compared to the saline treated control. EAC solid tumour was insensitive to saffron extract treatment since it failed to inhibit the growth of solid tumour (Figure 2). Oral administration of 100 mg/kg of saffron extract significantly (P<O.OOl) inhibited the growth of sarcoma-180 solid tumour in mice by 41% as compared to control mice (Figure 3).
_i)_ 1 OOmg/kg, 1-9 days
150mg/kg,9-17 days
0 1 0 20 30 40
DAYS AFTER TUMOUR INOCULUM
Fig. 1. Inhibitory effects of saffron extract on the growth of Daltons Lymphoma (DLA) solid tu- mour. (A) Saline treated control, (0) mice which were treated with an oral dose of 100 mgkg, (0 ) 50 mg/kg of saffron extract daily for nine days, and (0) 150 mgkg, nine days after the tumour inoculum. One million cells were s.c inoculated into the hind leg of mice to induce DLA solid tumour. Significance ***P<O.001, * P<O.O1
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S.C. NAIR ET AL.
0 10 20 30 40
DAYS AFTER TUMOUR INOCULUM Fig. 2. Inhibitory effects of saffron extract on the growth of Ehrlich ascites carcinoma (EAC) solid
tumour. (0) Saline treated control and (.) mice treated with 100 mg/kg of saffron extract daily for nine days. One million cells were inoculated S.C. into the hind leg of mice to induce EAC solid tumour. Significance * P z 0.05.
Serum p-Carotene and Vitamin A levels in mice Oral administration of saffron extract for nine days to the sarcoma-I80 solid tumour bearing mice elevated the serum p-carotene and vitamin A levels as compared to the respective tumour and non-tumour bearing controls treated with saline. A dose of 100 mg/kg of saffron extract significantly (PcO.001) increased the serum p-carotene and vitamin A levels on the tenth day. However the eleva- tions in serum p-carotene and vitamin A levels were reduced after withdrawing treatment, as noted on the 30th day (Table 2). Administration of 50 mg/kg of saffron extract relatively enhanced serum vitamin A but not p-carotene levels. The data also showed that the treated mice (100 mg/kg) maintained increased serum levels up to 30 days while the p-carotene values were near normal in mice which were treated with 50 mg/kg of saffron extract.
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SAFFRON EFFECTS ON VITAMIN A AND ANTITUMOUR ACTIVITY 111
5
4
0 0 5 10 15 2 0 2 5 3 0 3 5 40
DAYS AFTER TUMOUR INOCULUM
Fig. 3. Inhibitory effects of saffron extract on the growth of sarcoma-I80 (S-180) solid tumour. (0) Saline treated control, (0) mice which were treated with a oral dose of 100 mgkg and (0 ) 50 mgkg of saffron extract daily for nine days. One million cells were s.c inoculated into the hind leg of mice to induce S-180 solid tumour. Significance ***P<O.OOI, * P<O.OI.
DISCUSSION
Epidemiological studies have pointed to a protective role of dietary factors in the etiology of cancer of breast, ovary, lung and oral cavity (Boone et al., 1990). The extracts of spices are reported to possess tumour growth inhibitory and chemo- preventive substances (Nagabushan and Bhide, 1987). Saffron extract, a com- monly used food spice, along with other extracts of Nigella sativa, Ixora javani- ca and Saraka asoca, is a principle constituent of a preparation used for the treatment of cancer by the Indian system of medicine (Nair et al., 1991b; Pan- ikkar et al., 1986).
Our earlier studies showed the cytotoxic, antipromoting and antimutagenic activity of saffron extract (Salomi et al., 1991b) and its antitumour activity
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Table 2. Estimatian of serum vitamin A from Sarcoma-I80 tumour bearing albino mice treated with or without saffron extract .
Groups/ Tumour P-carotene Vitamin A Treatment pg/lOO ml serum pg/IOO ml serum
Day 10 Day30 Day 10 Day30
Normal (A) saline
0.78f0.12 ND 1 1.26f2.18 ND (100 p.l, 1X9days) -
Control (B) saline (100 pl, 1X9days) S-180(1x106cells) 0.82f0.09 ND 12.61f2.05 ND
Treated (C) (saffron extract, 50 mg/kg, 1x9 days) S-180 (1x106 cells) 1.59f0.35* 1.3f0.28 20.1rtl.l** 14.32f2.04
Treated (D) (saffron extract, 100 mg/kg, 1x9 days) S-180 (1x106 cells) 2.of0.31** 1.53rt0.1 28.7f5.9** 20.97f1.58
All the mice were inoculated (s.c.) with lo6 sarcoma-180 tumour cells on day 0 and 24 h later treated with saffron extract, orally, once a day for nine days using a gavage. Vitamin A estimations were done by sacrificing ten mice from each group on days 10 and 30, respectively. Values represented are Meanf SD from three separate experiments using 20 mice/group/experiment. ND indicates not done. * P<0.02, **P<O.OOl
against ascites tumours in mice (Nair et al., 1991a). Toxicity studies indicated the absence of severe toxicological manifestations in the liver, kidney, or bladder as confirmed by both enzyme and pathological studies (Nair et al., 1991a). We also noted that saffron extract at a dose of 50 mg/kg, i.p., could effectively modulate the antitumour activity of cisplatin in mice (Nair et al., 1991d). Re- cently we have reported that saffron enhanced the glutathione and related en- zyme levels in cultured cells and induced proliferation of thymocytes in mice (Nair et al., 1992).
The present chromium release cytotoxicity studies confirmed the cytolytic potential of saffron extract in vitro and the sensitivity of DLA and S-180 tumour cells to the extract; this observation was in accordance with our results from cytotoxicity studies (Nair et al., 1991a). Thymidine incorporation assay indicat- ed the action of the extract could be monitored by DNA synthesis. Antitumour studies showed the tumour growth inhibitory effects on subcutaneously trans- planted solid tumour, the preferential sensitivity of DLA and S-180 tumour to saffron extract as compared with EAC tumour. This could possibly be due to variable receptor patterns exhibited by these tumour cells. The elevations of
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SAFFRON EFFECTS ON VITAMIN A AND ANTITUMOUR ACTIVITY 113
serum p-carotene and vitamin A levels in sarcoma tumour bearing mice after the administration of saffron extract is interesting, since saffron is found to contain water-soluble carotenoids (Pfander and Witwer, 1975). Many reports are availa- ble on the role of p-carotene in reversing preneoplastic lesions in both humans and rodents (Schwartz and Skhlar, 1988). Chemical analysis using UV, IR, NMR and MS of the antitumour compound from saffron extract indicated that crocin (a naturally occurring carotenoid) may be the active principle responsible for the observed anticancer effects (manuscript communicated). The mechanism by which the naturally occurring carotenoid, crocin, exerts its antitumour effect may be by the metabolic conversion of carotenoids to retinoids in mice (Nair et al., 1992). It remains to be investigated whether crocin inhibits tumourigenesis as a free radi- cal scavenging agent, a direct antioxidant or by modulating the functional levels of other antioxidants.
ACKNOWLEDGEMENT
The authors wish to thank Indian Council Medical Research for financial assistance and Drs. Sohaib A. Khan (University of Cincinnati, Ohio, USA) and K. Saggor (University of Minnesota) for the chemical analysis of the extract, Dr. Madhavan Pillai (Department of Applied Chemistry, Cochin University) for the interpretation on the chemical analysis and Dr. K. Shivaprasad (Dept. Surgery, Amala Hospital) for the valuable critical discussions.
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Accepted May 14, 1993
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