Cancer Letters, 63 (1992) 41-46 Elsevier Scientific Publishers Ireland Ltd.
principles from Nigella satiua seeds
Amala Nagor, P.O.,
1 August 1991) 13 January 1992)
family, the seeds of which are used as a condiment in curries and with other aromatic substances and bitters. These seeds are ascribed to have many medicinal properties in traditional medicine [l]. Dhar et al. (1968) [Z] have reported on the various biological activities including anti-neoplastic activity of N. satiua seeds. Since then, various authors have undertaken studies on antibacterial, antifungal and antihelminthic effects of N. satiua seeds [3,4]. Further studies on the antitumour activity of N. satiua seeds are not recorded. Preliminary experiments with methanolic extract of N. satiua seeds indicated the presence of an antitumour principle . In this paper we are presenting the results of in vitro and in vivo antitumour experiments carried out using the active principle isolated from N. satiua seeds.
The actiue principle of Nigella satiua seeds containing certain fatty acids was studied for antitumour actiuities against Ehrlich as&es carcinoma (EAC), Dalton’s lymphonia ascites (DLA) and Sarcoma-180 (S-180) cells. In uitro cytotoxic studies showed 50% cytotoxicity to Ehrlich ascites carcinoma, Dalton’s lymphoma ascites and Sarcoma-180 cells at a concentration of 1.5 pg, 3 pg and 1.5 pg respectiuely with little activity against lymphocytes. The cell growth of KB cells in culture was inhibited by the actiue principle while K-562 cells resumed near control values on day 2 and day 3. Tritiated thymidine incorporation studies indicated the possible action of an active principle at DNA level. In uiuo EAC tumour development was completely inhibited by the active principle at the dose of 2 mg/mouse per day x 10.
Nigella satiua seeds were purchased from local market which were under storage for a period of less than 2 months and authenticated by an experienced botanist. Seeds were cleaned, dried, powdered and kept at 4OC.
Keywords: antitumour; Nigella satiua; Ehrlich ascites carcinoma; Dalton’s lymphoma ascites; Sarcoma- 180 Introduction
Isolation of the antitumour principle The antitumour principle was isolated from a methanolic extract of N. satiua seeds as described by us earlier . Briefly, the seed powder was extracted with methanol, passed through a silica gel column and eluted with the solvent system of methanol:water (9:l). The
Nigella satiua, commonly known as black cumin is a dicotyledon of the Ranunculacea Correspondence Centre, Amala
for K.R. Panikkar, Nagar P.0, Thrissur
0304-3835/92/$05.00 Printed and Published
Amala Cancer Research 600 553, Kerala, India.
0 1992 Elsevier Scientific Publishers in Ireland
active fraction was collected and further separated by ascending paper chromatography using chloroform as the solvent system. The active compound was eluted with methanol, evaporated under reduced pressure to render the product alcohol free and stored at 4OC. At each stage of purification the activity was ascertained using in vitro cytotoxic experiments. The active principle was dissolved in ethanol. The appropriate concentration of sample was diluted with PBS and used for in vitro and in vivo experiments. During dilution the amount of alcohol in the vehicle was brought below 2%. For all the experiments controls were maintained with equal concentration of alcohol in the same schedule.
Cytotoxicity was monitored using Dalton’s lymphoma ascites cells, Ehrlich ascites carcinoma cells and Sarcoma-180 cells propagated intraperitoneally in Swiss albino mice. Various concentrations (0.5 fig - 50 pg) of active principle was incubated with 1 x lo6 cells at 37OC for 3 h in 1 ml PBS. The percentage of dead cells was determined using trypan blue exclusion method. The amount of sample needed to produce 50% cytotoxicity was derived from a graph by plotting drug concentration against the percentage of dead cells. Cytotoxicity
KB-cells (oral carcinoma cells of man) and K-562 (human myelogenous leukemia cells) were used to determine the cytotoxicity in cultured cells. Nearly 0.25 million KB cells maintained in minimum essential medium containing 10% foetal calf serum was incubated, at 37OC and after 24 h, aliquots of the active principle (0.2 pg/ml- 5 pg/ml) were added. All the bottles were kept in triplicate. After 5 days, the cells were removed by trypsinization, stained with trypan blue and live cells were counted using a hemocytometer. K-562 cells (1 x 106) cultured in RPM1 supplemented with 10% foetal calf serum was
incubated with 10 pg/ml of active principle at 37OC. The number of cells were counted from 1st day onwards for 3 consecutive days. Cytotoxicity
The ficoll-hypaque procedure was used for the isolation of lymphocytes from the blood of acute lymphocytic leukemia patients. These cells, after washing with PBS, were treated with the active principle at various concentrations (100 - 500 pg) and incubated for 3 h at 37°C. Determination
The effect of active principle on the incorporation of tritiated thymidine into DNA was done by incubating DLA cells (1 x 106) with 2 &i of [3H]thymidine in the presence of various concentrations of the drug (0.0 1 - 5 vg) at 37OC for 4 h. After incubation, DNA was precipitated with 0.8 M perchloric acid at 4OC and redissolved in 0.5 N NaOH and counted in a liquid scintillation counter, after the addition of a dioxan based cocktail. In vivo studies
In vivo studies were carried out in Swiss albino mice 8 - 10 weeks old and 20 - 22 g in weight, using DLA cells and EAC-cells. Three groups of Swiss albino mice were transplanted i.p. with 1 x lo6 cells (DLA or Ehrlich). After 24 h of cell transplantation, the active principle at a concentration of 100 mg/kg body wt. and 50 mg/kg body wt. was administered i.p. to first and second groups of mice, respectively. This was continued for 10 days. The 3rd group was the controls which received the vehicle only. The death rate of animals was observed and increase in life span was calculated using the formula (T-C)/C x 100 where T is the average life span of test animals and C, the average life span of control animals. Solid tumours were produced in mice by injecting 1 x lo6 DLA cells subcutaneously on hind limbs. The active principle (25 mg/kg body wt.) was injected subcutaneously at the same site on the following day and continued for 10 days. Solid tumours were measured
from day 6 and the volume was calculated using the formula, tumour volume V = 4/3 a r12r2where r2 is the major radius . Results The in vitro cytotoxic effect of the active principle is shown in Table I. The minimum concentration of active principle needed for 50% cytotoxicity was 1.5 pg/ml for EAC cells and S-180 cells while this value was 3.0 pg/ml for DLA cells. The active principle was found to inhibit the growth of KB cells in culture. For a concentration of 0.5 pg/ml, 60% inhibition of cell growth was obtained (Fig. 1). In K-562 culture experiments, there was a temporary retardation of cell growth on day 1 (i.e. after 24 h) which resumed the near control values on day 2 and day 3 (Fig. 2). These values were obtained at a concentration of 10 pg/ml of the active principle. It was found that the active principle caused 50% cytotoxicity to lymphocytes at a concentration of 0.28 mg/ml (Fig. 3). This was 186-times higher than the concentration needed for 50% cytotoxicity of tumour cells. The effect of active principle on the thymidine uptake by DLA-cells was determined (Fig. 4). Additions OF active principle drastically reduced the thymidine uptake and the concenTable 1. In vitro cytotoxicity different cell lines. Cell lines used
Concentration of active principles required for 50% cytotoxicity pg/m)
DLA-cells EAC-cells S-HO-cells
3.0 (2.5 -4.0) 1.5 (0.5-2.0) 1.5 (1.0-3.0)
Values are expressed as the amount of active principle needed in micrograms to produce 50% cytotoxicity in 3 h at 37°C. Values are the mean of 3 separate determinations. The amount of active principle needed for 50% cytotoxicity was determined from a graph plotting the concentration against the percentage of dead cells.
Fig. 1. Cytotoxicity of active principle on KB-cell culture. KB cells were incubated at 37’C and after 24 h different concentrations of active principle were added. After 5 days of incubation the number of live ceils were counted and the % of live cells in experimental animals was calculated with respect to the number of live cells in controls. Values are the mean of 3 separate experiments. C-control; 1, active principle 0.2 pg/ml; 2, active principle 0.5 pg/ml; 3, active principle 1.0 pg/ml; 4, active principle 2.0 &ml; 5, active principle 5.0 pg/ml. Ethanol used to dissolve the active principle by itself did not have any effect on cell growth at concentrations studied here.
tration of active principle needed for 50% inhibition was 0.1 pg/ml. Studies on the effect of active principle on tumour development was also monitored. It was found that 100 mg/kg body wt. of active principle could inhibit the ascites tumour development of EAC cells and all animals remained normal without any tumour (Table II). In experimental animals treated with 50 mg/kg body wt. of active principle, a significant (P < 0.001) increase in average life span was obtained and the % ILS was 64.10. In the case of DLA cells the average life span of control animals was 24.38 f 4.96 days and 45.08 f 5.2 days (P < 0.001) and 34.3 f 4.5 (P < 0.001) for experimental animals treated with 100 mg/kg body wt. and 50 mg/kg body wt. of active principle respectively (Table 3). Solid
Fia. 2. Cytotoxicity of active principle on K-562 cell culture. K-562 cells were incubated with 10 pg/ml of active principle at 37’C. The number of cells were counted from 1st day onwards for 3 consecutive days. % of live cells in experimentals is calculated with respect to the number of live cells in controls. Ethanol used to dissolve the active principle by itself did not have any effect on cell growth at concentrations used here. Values are mean of three separate experiments. 1 - 0 h, 2 - 24 h, 3 - 48 h, 4-72 h.
of sample (kg)
Fig. 4. Effect of active principle on [3H]thymidine incorporation into DLA cells. 50% inhibition in the incorporation of 3-h thymidine into the DNA of DLA cells was obtained at a concentration of 0.1 pg/ml.
tumour experiments with DLA cells were also done. The tumour volume of experimentals and controls were more or less similar on different days of observation (Fig. 5). Discussion
of sample (1~9)
Fi3. 3. In vitro cytotoxicity of active principle on isolated lymphocytes. Lymphocytes were isolated from the blood of acute lymphocytic leukemia (ALL) patient by the Ficoll-Hypaque method. Blood samples were collected prior to the commencement of chemotherapy. Ethanol used to dissolve the active principle did not produce any cytotoxicity at the concentrations studied.
The range of concentration of active principle required for 50% cytotoxicity of various cell lines in vitro was almost similar. Culture experiments showed that percentage growth inhibition of KB-cell culture was more significant than that of K-562 cells. In vivo studies showed that the active principle could completely inhibit the EAC-tumour development inspite of that, experiments with DLA-cells showed only partial prevention of tumour development. These results along with cytotoxic study on lymphocytes showed that the active principle was less active on lymphocyte derived cell lines. Such specificity of action of Ixora jauanica extract on different cell lines has already been reported . A thymidine incorporation study revealed the possible action of the active principle at DNA level.
45 Table II.
Effect of active principle
EAC-cells 1 x lo6
on development of animals
alive after 40 days
EAC-cells 1 x lo6 + active principle (50 mg/kg body wt.)
Increase in life span (ILS)“/;,
Average life span
EAC-cells 1 x lo6 + active principle (100 mg/kg body wt.)
(days) 19.0 f
All animals remained as normals without any tumour
Tumour was initiated by i.p. inoculation of 1 x 10 EAC cells. The active principle was administered i.p. on the following day at a concentration of 100 mg/50 mg per kg body wt. for 10 days. Values are the mean of 10 mice per group. P < 0.001.
The active principle isolated is a liquid substance which was used for antitumour studies. This active principle contained a long chain fatty acid, but there may be other compounds as well preferably with double bonds in Table III.
Effect of active principle
on development 40 days
view of the liquid nature of the active principle. A similar work carried out by Das (1989)  found several fatty acids as the active antitumour principle from Jawaharene (Aspergillus). The presence and position of double
and survival of animals
Average life span
Increase in life (ILS) %
(days) DLA-cells 1 x lo6
DLA-cells 1 x lo6 + active principle (10 mg/kg body wt.)
DLA-cells (1 x 106) + active principle (50 mg/kg body wt.)
Tumour was initiated by i.p. inoculation of 1 x lo6 DLA cells. The active principle was administered i.p. on the following day at a concentration of 100 mg/50 mg per kg body wt. for 10 days. Values are mean of 10 mice per group. lP < 0.001.
The authors are thankful to the Council of Scientific & Industrial Research, New Delhi, for awarding a Senior Research Fellowship to M.J. Salomi. The authors are also thankful to Dr. Madhavan Pillai, Professor of Chemistry, Cochin University of Science and Technology, Cochin for the identification of the fatty acid of the active principle.
v $ 2 b E 2
The effect of active principle on the development of a solid tumour by DLA cells in mice. Values are the average of 6 animals in each group. O-O, tumour volume of non-treated mice. O-O, tumour volume of treated mice.
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