Cancer Letters, 57 (1991) 109-114 Elsevier Scientific Publishers Ireland
109 Ltd.
activity of saffron (Crocus sativus)
Antitumour S.C.
Nair,
B. Pannikar
Amala
Cancer
Research
Centre,
(Received
4 December
1990)
(Accepted
28 January
1991)
and
K.R.
Amala
Nagar
Panikkar P.O.,
Trichur.
Summary
Antitumor activity ofsaffron (Crocus satiuus) extract a commonly used spice in India was studied against intraperitoneally transplanted sarcoma-l 80 (S-l BO), Ehrlich ascites Carcinoma [EAC) and Dalton’s lymphoma ascites (DLA) tumours in mice. Oral administration of 200 mg/kg body weight of the extract increased the life span of S-180, EAC, DLA tumour bearing mice to lll.O%, 83.5% and 112.5 W , respectively. The same extract was found to be cytotoxic to P38B, S-180, EAC and DLA tumour cells in vitro. Thymidine uptake studies indicated the mechanism of action of the extract at the site of DNA synthesis. Toxicity studies showed that the hematological and biochemical parameters were within normal range. These results indicate the potential use of saffron as an anticancer agent.
Keywords:
antitumour
activity; cytotoxicity;
saffron extract Introduction
Dietary factors play a significant role in both cancer promotion and prevention. Generally neoplastic change is associated with alterations in the vital cellular functions. Many comCorrespondence to: Dr. K.R. Panikkar. Amala Cancer Research Centre, Amala Nagar P.O. Trichur 680 553. Kerala. India.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific Publishers in lreland
Kerala
(India)
pounds from natural products have shown differentiation inducing activity thus useful in the treatment of cancer [ 11. Extracts of some spices were found to inhibit the growth of transplanted tumours in mice [Z] as well as being cytotoxic to cells in tissue culture [ 31. Curcumin, the colouring matter from turmeric inhibited the growth of forestomach tumours induced by benzo [a] pyrene [4] and skin carcinomas induced by 7,12-dimethylbenz [a] anthracene in mice [5]. We have recently reported on the antipromoting and nonmutagenic activity of saffron (Crocus satiuus) and black cumin (Nigella satiua) extracts, commonly used food spices in India [ 61. In this paper we investigated the antitumour activity of saffron extract against transplanted tumours in mice. Materials
and
Methods
Saffron (Crocus satiuus) used in this study was purchased from the Government Emporium, Kashmir, India and stored at 4OC till further use. Saffron was extracted with ethanol (95%) and purified by the method described by us earlier [ 71. Cytotoxicity assay in vitro Cytotoxicity was determined using Sarcoma 180 (S-180), P388 leukemia, Ehrlich ascites carcinoma (EAC) and Dalton’s lymphoma ascites (DLA) tumour cells [8]. S-180, P388 and EAC was obtained from the Cancer Ireland Ltd
110
Research Centre, Calcutta. All the tumour cells were individually maintained in male Swiss albino mice. (P38 leukemia maintained in DBA/Z mice), by injection of 1 x lo6 in 1 ml sterile saline i.p. After visible tumours appeared the cells were aspirated, washed 3 times with phosphate buffered saline (PBS, pH 7.4). Cells (1 x lo6 were incubated with various concentrations of the drug in PBS for 3 h at 37OC. After incubation the percentage of live cells was determined by the trypan blue exclusion method. The drug needed to produce 50% cytotoxicity to cells was obtained by plotting a graph of drug concentrations (pg/ml) against percentage of live cells with respect to control (without drug) after assay. Normal Balb/c mouse spleen cells (1 x 106/ml MEM) (1 ml) was also subjected to cytotoxic studies as above in presence of the drug at different concentrations. About 3-5s cell death was observed in the control (without drug). Effect of the drug on DNA biosynthesis Incorporation of tritiated thymidine (B.A.R.C., Bombay) into the cellular DNA of P388 leukemia cells was conducted to ascertain the effect of the drug on DNA biosynthesis. 2 ml cell suspension (1 x lo6 cells/ml MEM) containing 10% goat serum were added to the tubes with appropriate drug concentrations. Tritiated thymidine (3H-Tdr) at a final concentration of 2 PCi was added to the cell suspension. The tubes were shaken gently to ensure even distribution of the drug and incubated at 37OC for 4 h. After incubation DNA was precipitated with 0.8 M perchloric acid at 4OC and dissolved in 0.5 N NaOH and counted in a liquid scintillation counter (LKS Rackbeta 1209) [9]. Effect of the drug on the growth of ascites tumours in mice Inbred male Swiss albino mice weighing 18-20 g, 9- 11 weeks old were used for this study. The mice were maintained on standard mouse feed. The mice were randomized into 6 groups, containing 7 or 8 mice per group
as indicated. Four milligrams of the drug were dissolved in 1 ml sterile saline equivalent to 200 mg/kg body weight. Groups A, C and E received 1 x lo6 Sarcoma-180, Ehrlich ascites carcinoma and Dalton’s lymphoma tumour cells in 1 ml sterile saline i.p., respectively. These groups were untreated to be used as the controls. Groups B. D and F received tumour cells as above, and the drug was administered at a dose of 200 mg/kg orally once per day, using a day 1-9 regimen, 1 day after the tumour inoculum. The survival time of each of the mice was noted [ 81. Toxicological studies Peripheral blood for hematological and biochemical studies to assess the toxicity of saffron extract was collected from the caudal veins of mice after 9 days of treatment and values compared with that of control (saline treated) mice. Total WBC counts were performed using a hematocytometer while serum glutamate pyruvate transaminase (SGPT) and serum alkaline phosphatase (SAKP) estimated by the 2,4-dinitrophenylhydrazine and 4-amino antipyrine method, respectively. Blood urea nitrogen was estimated by diacetyl monoxime method. Results
Cytotoxic studies There was a dose dependent cytotoxicity of tumour cells (Fig. 1). The concentration of the drug needed to produce 50% cytotoxicity of Sarcoma 180 (S-180)) P388 leukemia, Ehrlich ascites carcinoma (EAC) and Dalton’s lymphoma (DLA) tumour cells was 28, 30, 17 and 9 pg/ml, respectively. Normal mouse spleen cells were insensitive to the drug even at higher concentrations. lnhibition of DNA biosynthesis The studies on the effect of the purified saffron extract on DNA biosynthesis of P388 leukemia cells showed a concentrationdependent decrease in thymidine incorporation (Table I). The concentration of the drug
111
S-180, EAC and DLA ascites tumours in mice. All the tumours responded well to saffron extract treatment. A significant increase in the life span of S-180, EAC and DLA ascites tumourbearing mice treated with saffron extract was observed (Table II). The increase in the life span was nearly 2-3-fold as compared with untreated (control) the tumour-bearing animals. The lethal dose (LD,,) required to kill 50% of the animals receiving saffron extract was >600 mg/kg body weight.
20
Concentration
60
100
80
of drug (~g/mi)
Fig. 1. Cytotoxicity of saffron extract to tumour cells in vitro; 1 x lo6 cells (0) P388, (0) S-180, (B), EAC ( q ) DLA, were incubated with or without saffron extract for 3 h at 37OC. Viability determined by trypan blue exclusion method. Spleen cells ( o ) were insensitive to the extract. Values are mean + S.D. from 3 studies. Significance P < 0.001.
needed to produce thymidine incorporation Antitumour
50% inhibition was 4.2 pg/ml.
in
studies
Administration of 200 mg/kg of saffron extract orally by gavage inhibited the growth of Table 1. Effect of saffron extract on DNA biosynthesis Concentration the drug
of
Cpm
f
mean
Toxicity studies Hematological and biochemical studies to ascertain drug-induced toxicities indicated that SGPT, SAKP and blood urea nitrogen levels were maintained within the normal range. A significant decrease in hemoglobin levels in the saffron extract-treated group was noted as compared to saline treated (control) animals. However WBC levels was found to be normal (Table III). Discussion
Extracts of spices are found to possess growth inhibiting substances [ 21. The current studies indicated the cytotoxic action of saffron extract on a variety of tumour cells in vitro. S-180, EAC and DLA tumour cells were sensitive to the extract at lower concentrations while normal mouse spleen cells were
of P388 murine leukemia
S.D.
cells
Percent incorporation =t mean S.D.
Percent inhibition * mean S.D.
(ccg/mI) 0
(Control) 1 2.5 5.0 10 25 50% IC (inhibition in incorporation) 4 observations. All values showed
36530
f
1683.5
27477 23916 15529 12162 2637
zt 361.2 + 756.6 + 586.7 zt 1260.8 zt 179.3
was obtained at a concentration significance P < 0.001.
75.21 65.67 42.50 33.29 7.2
iz zt f. + f
0.985 2.36 1.6 3.45 0.50
of 4.2 pg/ml.
24.77 34.32 57.48 66.7 92.79
zt + iz zt *
0.985 2.36 1.6 3.45 0.50
Values are cpm + mean S.D. from
4/8
8/S
B/B 8/8
oral l-9 -
200 mg/kg oral 1-9 200 mg/kg oral l-9
7/7
200 mg/kg
days
days
days
2/7
8/8
6/8
8/8
O/8
6/7
o/7
6/8
O/8
4/8
O/8
4/7
o/7
Z/8
O/8
O/8
O/8
o/7
o/7
60 Days
35 Days
15 Days
25 Days
after tumour
No. of mice survived inoculation
-
Dosage and mode of therapy
in mice
58.27
27.42
32.29
17.59
40.88
19.35
1.82
2.22
f
5.54”’
+ 2.92
zt 3.89”
f
+z 0.08’
l
Mean survival time (MST) =t mean S.D.
l
*
Significance
l
values ( * ‘P < 0.001)
(’ lP < 0.01).
ILS = increase of life span = T-C/C x 100 (T = mean survival time of treated mice, C = mean survival time of (untreated) Values are mean f S.D. from three experiments using 7 or 8 mice per group/experiment as inciated.
F
E
D
C
Ehrlich ascites carcinoma (control) Ehrlich ascites carcinoma (treated) Dalton’s lymphoma ascites (control) Dalton’s lymphoma ascites (treated)
Sarcoma-180 (control) Sarcoma-180 (treated)
A
B
Tumor model
Effect of saffron extract on the growth of ascites tumours
Groups
Table II.
control mice.
112.5
83.5
111.0
ILS (9%)
113
Table 111. Haematological extracts daily for 9 days.
and biochemical
of mcie treated with a dose (200 mg/kg
Haematological level (g%)
Blood urea nitrogen (mg/ 100 m.l serum)
432
15.8 zt 0.8
18.57
f
130
11.7
f
18.57
* 1.8
Treatment and dose
Total WBC (counts/mm3)
Control (saline-treated) mice) Saffron extract 200 mg/kg (oral) Significance
9400
f
8350
f
109 Ltd.
activity of saffron (Crocus sativus)
Antitumour S.C.
Nair,
B. Pannikar
Amala
Cancer
Research
Centre,
(Received
4 December
1990)
(Accepted
28 January
1991)
and
K.R.
Amala
Nagar
Panikkar P.O.,
Trichur.
Summary
Antitumor activity ofsaffron (Crocus satiuus) extract a commonly used spice in India was studied against intraperitoneally transplanted sarcoma-l 80 (S-l BO), Ehrlich ascites Carcinoma [EAC) and Dalton’s lymphoma ascites (DLA) tumours in mice. Oral administration of 200 mg/kg body weight of the extract increased the life span of S-180, EAC, DLA tumour bearing mice to lll.O%, 83.5% and 112.5 W , respectively. The same extract was found to be cytotoxic to P38B, S-180, EAC and DLA tumour cells in vitro. Thymidine uptake studies indicated the mechanism of action of the extract at the site of DNA synthesis. Toxicity studies showed that the hematological and biochemical parameters were within normal range. These results indicate the potential use of saffron as an anticancer agent.
Keywords:
antitumour
activity; cytotoxicity;
saffron extract Introduction
Dietary factors play a significant role in both cancer promotion and prevention. Generally neoplastic change is associated with alterations in the vital cellular functions. Many comCorrespondence to: Dr. K.R. Panikkar. Amala Cancer Research Centre, Amala Nagar P.O. Trichur 680 553. Kerala. India.
0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific Publishers in lreland
Kerala
(India)
pounds from natural products have shown differentiation inducing activity thus useful in the treatment of cancer [ 11. Extracts of some spices were found to inhibit the growth of transplanted tumours in mice [Z] as well as being cytotoxic to cells in tissue culture [ 31. Curcumin, the colouring matter from turmeric inhibited the growth of forestomach tumours induced by benzo [a] pyrene [4] and skin carcinomas induced by 7,12-dimethylbenz [a] anthracene in mice [5]. We have recently reported on the antipromoting and nonmutagenic activity of saffron (Crocus satiuus) and black cumin (Nigella satiua) extracts, commonly used food spices in India [ 61. In this paper we investigated the antitumour activity of saffron extract against transplanted tumours in mice. Materials
and
Methods
Saffron (Crocus satiuus) used in this study was purchased from the Government Emporium, Kashmir, India and stored at 4OC till further use. Saffron was extracted with ethanol (95%) and purified by the method described by us earlier [ 71. Cytotoxicity assay in vitro Cytotoxicity was determined using Sarcoma 180 (S-180), P388 leukemia, Ehrlich ascites carcinoma (EAC) and Dalton’s lymphoma ascites (DLA) tumour cells [8]. S-180, P388 and EAC was obtained from the Cancer Ireland Ltd
110
Research Centre, Calcutta. All the tumour cells were individually maintained in male Swiss albino mice. (P38 leukemia maintained in DBA/Z mice), by injection of 1 x lo6 in 1 ml sterile saline i.p. After visible tumours appeared the cells were aspirated, washed 3 times with phosphate buffered saline (PBS, pH 7.4). Cells (1 x lo6 were incubated with various concentrations of the drug in PBS for 3 h at 37OC. After incubation the percentage of live cells was determined by the trypan blue exclusion method. The drug needed to produce 50% cytotoxicity to cells was obtained by plotting a graph of drug concentrations (pg/ml) against percentage of live cells with respect to control (without drug) after assay. Normal Balb/c mouse spleen cells (1 x 106/ml MEM) (1 ml) was also subjected to cytotoxic studies as above in presence of the drug at different concentrations. About 3-5s cell death was observed in the control (without drug). Effect of the drug on DNA biosynthesis Incorporation of tritiated thymidine (B.A.R.C., Bombay) into the cellular DNA of P388 leukemia cells was conducted to ascertain the effect of the drug on DNA biosynthesis. 2 ml cell suspension (1 x lo6 cells/ml MEM) containing 10% goat serum were added to the tubes with appropriate drug concentrations. Tritiated thymidine (3H-Tdr) at a final concentration of 2 PCi was added to the cell suspension. The tubes were shaken gently to ensure even distribution of the drug and incubated at 37OC for 4 h. After incubation DNA was precipitated with 0.8 M perchloric acid at 4OC and dissolved in 0.5 N NaOH and counted in a liquid scintillation counter (LKS Rackbeta 1209) [9]. Effect of the drug on the growth of ascites tumours in mice Inbred male Swiss albino mice weighing 18-20 g, 9- 11 weeks old were used for this study. The mice were maintained on standard mouse feed. The mice were randomized into 6 groups, containing 7 or 8 mice per group
as indicated. Four milligrams of the drug were dissolved in 1 ml sterile saline equivalent to 200 mg/kg body weight. Groups A, C and E received 1 x lo6 Sarcoma-180, Ehrlich ascites carcinoma and Dalton’s lymphoma tumour cells in 1 ml sterile saline i.p., respectively. These groups were untreated to be used as the controls. Groups B. D and F received tumour cells as above, and the drug was administered at a dose of 200 mg/kg orally once per day, using a day 1-9 regimen, 1 day after the tumour inoculum. The survival time of each of the mice was noted [ 81. Toxicological studies Peripheral blood for hematological and biochemical studies to assess the toxicity of saffron extract was collected from the caudal veins of mice after 9 days of treatment and values compared with that of control (saline treated) mice. Total WBC counts were performed using a hematocytometer while serum glutamate pyruvate transaminase (SGPT) and serum alkaline phosphatase (SAKP) estimated by the 2,4-dinitrophenylhydrazine and 4-amino antipyrine method, respectively. Blood urea nitrogen was estimated by diacetyl monoxime method. Results
Cytotoxic studies There was a dose dependent cytotoxicity of tumour cells (Fig. 1). The concentration of the drug needed to produce 50% cytotoxicity of Sarcoma 180 (S-180)) P388 leukemia, Ehrlich ascites carcinoma (EAC) and Dalton’s lymphoma (DLA) tumour cells was 28, 30, 17 and 9 pg/ml, respectively. Normal mouse spleen cells were insensitive to the drug even at higher concentrations. lnhibition of DNA biosynthesis The studies on the effect of the purified saffron extract on DNA biosynthesis of P388 leukemia cells showed a concentrationdependent decrease in thymidine incorporation (Table I). The concentration of the drug
111
S-180, EAC and DLA ascites tumours in mice. All the tumours responded well to saffron extract treatment. A significant increase in the life span of S-180, EAC and DLA ascites tumourbearing mice treated with saffron extract was observed (Table II). The increase in the life span was nearly 2-3-fold as compared with untreated (control) the tumour-bearing animals. The lethal dose (LD,,) required to kill 50% of the animals receiving saffron extract was >600 mg/kg body weight.
20
Concentration
60
100
80
of drug (~g/mi)
Fig. 1. Cytotoxicity of saffron extract to tumour cells in vitro; 1 x lo6 cells (0) P388, (0) S-180, (B), EAC ( q ) DLA, were incubated with or without saffron extract for 3 h at 37OC. Viability determined by trypan blue exclusion method. Spleen cells ( o ) were insensitive to the extract. Values are mean + S.D. from 3 studies. Significance P < 0.001.
needed to produce thymidine incorporation Antitumour
50% inhibition was 4.2 pg/ml.
in
studies
Administration of 200 mg/kg of saffron extract orally by gavage inhibited the growth of Table 1. Effect of saffron extract on DNA biosynthesis Concentration the drug
of
Cpm
f
mean
Toxicity studies Hematological and biochemical studies to ascertain drug-induced toxicities indicated that SGPT, SAKP and blood urea nitrogen levels were maintained within the normal range. A significant decrease in hemoglobin levels in the saffron extract-treated group was noted as compared to saline treated (control) animals. However WBC levels was found to be normal (Table III). Discussion
Extracts of spices are found to possess growth inhibiting substances [ 21. The current studies indicated the cytotoxic action of saffron extract on a variety of tumour cells in vitro. S-180, EAC and DLA tumour cells were sensitive to the extract at lower concentrations while normal mouse spleen cells were
of P388 murine leukemia
S.D.
cells
Percent incorporation =t mean S.D.
Percent inhibition * mean S.D.
(ccg/mI) 0
(Control) 1 2.5 5.0 10 25 50% IC (inhibition in incorporation) 4 observations. All values showed
36530
f
1683.5
27477 23916 15529 12162 2637
zt 361.2 + 756.6 + 586.7 zt 1260.8 zt 179.3
was obtained at a concentration significance P < 0.001.
75.21 65.67 42.50 33.29 7.2
iz zt f. + f
0.985 2.36 1.6 3.45 0.50
of 4.2 pg/ml.
24.77 34.32 57.48 66.7 92.79
zt + iz zt *
0.985 2.36 1.6 3.45 0.50
Values are cpm + mean S.D. from
4/8
8/S
B/B 8/8
oral l-9 -
200 mg/kg oral 1-9 200 mg/kg oral l-9
7/7
200 mg/kg
days
days
days
2/7
8/8
6/8
8/8
O/8
6/7
o/7
6/8
O/8
4/8
O/8
4/7
o/7
Z/8
O/8
O/8
O/8
o/7
o/7
60 Days
35 Days
15 Days
25 Days
after tumour
No. of mice survived inoculation
-
Dosage and mode of therapy
in mice
58.27
27.42
32.29
17.59
40.88
19.35
1.82
2.22
f
5.54”’
+ 2.92
zt 3.89”
f
+z 0.08’
l
Mean survival time (MST) =t mean S.D.
l
*
Significance
l
values ( * ‘P < 0.001)
(’ lP < 0.01).
ILS = increase of life span = T-C/C x 100 (T = mean survival time of treated mice, C = mean survival time of (untreated) Values are mean f S.D. from three experiments using 7 or 8 mice per group/experiment as inciated.
F
E
D
C
Ehrlich ascites carcinoma (control) Ehrlich ascites carcinoma (treated) Dalton’s lymphoma ascites (control) Dalton’s lymphoma ascites (treated)
Sarcoma-180 (control) Sarcoma-180 (treated)
A
B
Tumor model
Effect of saffron extract on the growth of ascites tumours
Groups
Table II.
control mice.
112.5
83.5
111.0
ILS (9%)
113
Table 111. Haematological extracts daily for 9 days.
and biochemical
of mcie treated with a dose (200 mg/kg
Haematological level (g%)
Blood urea nitrogen (mg/ 100 m.l serum)
432
15.8 zt 0.8
18.57
f
130
11.7
f
18.57
* 1.8
Treatment and dose
Total WBC (counts/mm3)
Control (saline-treated) mice) Saffron extract 200 mg/kg (oral) Significance
9400
f
8350
f
