Journd of Ethnopharmacology, Elsevier

Scientific

Publishers

31 (1991) 75-83 Ireland Ltd.

75

MODULATORY EFFECTS OF CROCUS SATIVUS AND NIGELLA SATIVA EXTRACTS ON CISPLATIN-INDUCED TOXICITY IN MICE

SATISH Amala

C. NAIR,

M.J. SALOMI,

BEENA

Cancer Research Centre, Amala

jAccepted

PANIKKAR

Nagar,

AND K.R. PANIKKAR

Trichur 680-553, Kerala IIndia,

July 30. 1990)

Summary

In mice, an extract of Crocus sativus stigmas partially prevented the decreases in body weight, hemoglobin levels and leucocyte counts caused by 2 mg/kg of cisplatin i.p. for 5 days. Treatment with the C. sativus extract also significantly prolonged the life span of cisplatin-treated mice almost three-fold. In contrast, an extract of Nigello sativa seed only tended to protect from cisplatin-induced falls in hemoglobin levels and leucocyte counts.

Introduction

Cisplatin (ck-diamine-dichloroplatinum) is an effective and widely used cytotoxic drug (Anonymous, 19821. Although renal toxicity is the dose-limiting factor, other associated toxicities include emesis, nausea, diarrhea, anorexia, hair epilation and myelosuppression (Allan et al., 19861.The associated renal toxicity can be overcome by methods of hydration during therapy (Comis, 19801. It is well established that the tumoricidal activity of cisplatin results at least in part from a reaction with DNA (Rosenberg, 19791. Other molecules, including RNA and proteins, also react with cisplatin (Howe-Grant and Lippard, 19801. These reactions occur either by a direct pathway in which the co-ordinated chlorides are displaced by nucleophilic entering groups or by an indirect pathway in which the entering nucleophiles react with cisplatin molecules that have already exchanged chloride groups for solvent (Hz01 (Reishus and Martin, 1961; Riley et al., 19821.Cisplatin protein interactions are important in determining the therapeutic efficacy of the antitumour agent. Binding of cisplatin to plasma proteins significantly alters the rate of clearance of the drug from circulation (Deconti et al., 1973; Bannister et al., 19771.Reactions of native proteins with cisplatin may be responCorrespondence to: Dr. K.R. Panikkar. Published

and Printed

in Ireland

sible for the observed toxicity to the kidneys and gastrointestinal tract (Slater et al., 1977; Borch and Pleasants, 19791. Considerable interest has been focused on the isolation of compounds that might be administered with cisplatin to reduce the dose-limiting toxicity (Allan et al., 1986). For these compounds to be useful, they must either be selectively absorbed by non-tumour cells or administered at an appropriate time, before or after the anti-tumour drug, when injury to tumour cells is irreversible and reversible to non-tumour cells e.g. MPG (Bmercaptopropionyl glycine) and WR-2721 (S-2-(8amino propylaminolethyl phosphorothioic acid) (Milas et al., 19841. Considering the above, this laboratory has investigated the effects of extracts of natural products, like Crocus sativus dried stigmas (saffron) and Nigellu sativa seeds, as potential protective agents against cisplatin-induced toxicity in Swiss albino mice. Materials and Methods

Drug and natural products Cisplatin was purchased from Biochem Pharmaceuticals Pvt. Ltd., Bombay. Saffron (Crocus sativus) was obtained from Government Emporium, Kashmir and NigeZZusativa seeds from a local market and stored at 2-4OC. Both plant materials were authenticated by C.D. Varghese, Botanist, St. Thomas College, Trichur, Kerala. All reagents used were of analytical grade. The dried seeds of Nigella sativa L. (Ranunculaceael were powdered and 5 g of the powder was extracted with 95O/6ethanol (100 ml1 thrice, overnight at 28OC with continuous stirring. The pooled extracts were evaporated under reduced pressure to a known volume (10 ml) and loaded on to a silica gel (Glaxo-India) column and eluted with 95% methanol/water (9 : 11. The active fraction (as indicated by brown colourl was collected and separated by ascending paper chromatography using chloroform as the solvent system. The active spot at the solvent front was cut, eluted with methanol (overnight), evaporated under reduced pressure to render the product alcohol-free and stored at 2OC. Approximate content of the active compound in the seeds was 2.2% (w/w). One gram of Saffron (Crocus sativus L., Irideae was extracted with 95% ethanol (50 ml), pooled and made to a known volume (5 ml) as above. Five millitres were loaded to a silica gel (Glaxo-India) column and eluted with petroleum benzene, chloroform, methanol and water, successively. The water fraction (yellow colourl containing the active ingredient was separated by ascending paper chromatography using butanohacetic acid/water (6 : 1 : 21 as the solvent system. The active spot (faint yellow colourl was cut, eluted with 95% methanol overnight, dried and stored as above. Approximate content of the active compound in the saffron was 5.6O/b(w/w). Schedule of drug administration Cisplatin (40 pgl was dissolved in 200 1.11 of sterile distilled water. The

77

extracts of saffron (1 mgl of N. sativa (1 mgl were reconstituted in 200 4 sterile physiological saline. The experiments were carried out on six groups of albino mice and each group consisted of seven animals. To the first group of mice, cisplatin was given 2 mg/kg i.p. for five alternate days. The second group was administered saffron extracts (50 mg/kg i.p.1 30 min before administering cisplatin (2 mg/kgl for five alternate days. The third group was given 50 mg/kg saffron extract as above but without cisplatin. In a similar way, the fourth group received N. sativu extract (50 mg/kg) and after 30 min cisplatin (2 mg/kg i.p.1 for five alternate days. Group five received only N. sat& extract (50 mg/kg i.p.1 for five alternate days. Group six was the control which received the same volume of normal saline for the same period. All the animals used were eight-week-old adult male Swiss albino mice weighting 22-23 g. They were maintained on standard mouse feed (Lipton, India). Biochemical and heamutological studies Peripheral blood for the various studies was collected from the caudal vein at different intervals. The total leucocyte counts were performed using a haemocytometer. Blood urea nitrogen (BUN) was estimated by the diacetyl monoxime method (Varley, 19761. Relative changes in the body weight was also noted. Results

Table 1 indicates the increase in life span (ILS) of treated mice. The mean survival time was 31.57 days for the cisplatin-treated group. With the C.

TABLE 1 EFFECT OF CROCUS SATIVUS AND NIGELLA OF CISPLATIN-TREATED SWISS MICE

SATIVA

EXTRACTS ON THE LIFE SPAN

Tabular values represent the mean f S.E.M. of 7 animals per group for three separate experiments W = 211. Treatment

Dose regimen

Survival time (days) mean f S.E.M.

Increase in life span (%l

P

Cisplatin

2 m03 i.p. x 5 days 2 mg/kg + 50 w3kg i.p. x 5 days 2 mg/kg+ 60 m&3 i.p. x 5 days

31.6 f 3.4

-

-

92.3 f 7.9

194

< 0.001

10

> 0.59

Cisplatin + C. sat&us Cisplatin + N. sativa

34.7 f

5.2

OF

CROCUS SATZVUS AND NZGELLA

S’ATZVA

EXTRACTS

ON THE BODY WEIGHTS

22.8 c 0.4

mice k3terile saline)

1.0

22.3 f

1.3

(50 m&kg) Normal Control

22.4 f

0.4

1.0

1.0

6Omg/kg) N. satiua

(50 mg/kg) Cisplatin (2 mg/kg) + N. sativa

60 mg/kg) C.satiuw

22.5 f

22.2 f

(2 mg/kg) + C. satin

22.1 f

(2 m&3) Cisplatin

Day 0

Body weight (g)

Cisplatin

Treatment

1.3

0.9

0.4

1.8

1.1

23.3 + 0.5

23.2 f

22.8 f

23.5 f

23.1 f

21.1 f

Day 3 1.2

0.2

23.4 f

0.4

22.8 zt 1.2

21.4 f

24.1 2 0.6

22.3 + 1.7

20.0 f

Day 5

23.5 f

22.7 f

19.9 f

24.1 f

21.7 f

19.3 f

Day 7

0.4

1.2

0.7

0.6

1.8

1.0

23.4 f

22.4 f

17.9 f

0.3

1.2

0.7

24.0 + 0.7

20.8 + 1.8

18.4 + 0.6

Day 10

Tabular values represent the mean k S.E.M. of 7 animals per group for three separate experiments.

EFFECT MICE

TABLE 2

6.5

+ 2.6

+ 0.3

- 20.2

+ 6.2

-

- 16.8

Maximum change (%I

-

> 0.1

< 0.001

>O.l

< 0.1

< 0.001

P

OF CISPLATIN-TREATED

SWISS

8066 f 162

11200 I 436

10206 f 748

10200 f 466

12206~826

9406 f 282

9200*260

9406 f 432

9006 * 614

9106 f 136

9206 f 403

Day 2

9000 f 310

DsYO

Tots1 leucocyte count kells/mma)

9350 + 47

9960+96

10006 f 210

10600+:786

10800 f 372

7206 f 196

Day 4

9466 & 179

9866 f 168

9866 f 71

10000~262

9600 f 302

7000 i- 142

Day 6

Statistically significant from normal control: lP < 0.05, **P < 0.01, ***P < 0.001.

Cisplatin !Z m&g) cisplatin (2 mg/kg) + c. wtivus (60 mg/kg) C. rativw (66 mg/kg) Cisplatia I2 mgikg) f N. rativa (50 mg/kg) N. sat&m (66 mg/kgl Normal control mice (sterile saline)

Treatment

9460 * 79 9806 * 392

9266 + 376

7866 f 130++

9460 f 68

8696 * 172

8360 f 112+

8606 f 76**

8400 f 116

9006 f 62

3666 f 67*++

Day 10

3866 * 218

Day 8

Values represent the mean * S.E.M. of seven mice per group for three separate experiments W = 21).

+l.l

+ 3.7

- 12.7

- 11.2

- 13.0

-61.2

Maximum change (I)

EFFECT OF CROCUS SATZVUS AND ZVZGEZLA SATZVA EXTRACTS ON THE TOTAL LEUCOCYTE COUNTS OF SWISS ALBINO MICE TREATED WITH CISPLATIN

TABLE 3

AND NlGELLA

SAZ’IVA EXTRACTS

12.5 + 0.9

12.0 f 0.7

13.4 rt 0.8

15.0 f 1.2

14.0 f 0.6

15.7 + 0.6

13.0 f 0.9

13.4 f 1.1

15.8 f 1.7

14.6 j: 1.4

15.9 j: 0.6

Day 2

13.9 f 1.6

Day 0

Hemoglobin Ievels (g%)

15.7 -t 1.0

12.6 + 0.9

13.8 2 1.3

12.0 -c 0.9

12.0 -c 0.9

11.7 + 1.1

Day 4

15.9 f 1.2

12.0 f 1.4

12.1 f 0.6

12.0 f 0.9

11.7 f 1.0

8.4 f 1.3

Day 6

Statistically significant from normal control: *P < 0.05, l*P < 0.01, ***P < 0.001.

Cisplatin (2 mgkg) Cisplatin (2 mgnitg)+ C. sativus 60 mg/kg) C.satin (50 mglkg) Cisplatin (2 mg/kg) + N. sativa 60 mglkgf N. sat& (50 mg/kg) Normal control mice &eriIe saline)

Treatment

15.7 * 0.9

10.9 f 0.9

12.0 + 0.7

11.7 f 0.6

10.9 f 0.9

7.5 k 0.7

Day 8

15.8 -c 0.8

10.8 r 0.4***

11.7 f 1.2*

11.7 -c 0.9**

10.0 rf: 0.4***

7.5 -c 0.6***

Day 10

- 0.6

- 26.0

- 25.9

- 12.6

- 23.1

- 46.0

Maximum change (Ok-11

ON THE HEMOGLOBIN LEVELS OF SWISS

Values represent the mean f S.E.M. of seven mice per group for three separate experiments (N = 21).

MODULATORY EFFECT OF CROCUS SATIVUS ALBINO MICE TREATED WITH CISPLATIN

TABLE4

81 TABLE 5 BLOOD UREA NITROGEN LEVELS OF SWISS ALBINO MICE RECEIVING CROCUS OR NIGELLA SATIVA EXTRACTS WITH OR WITHOUT CISPLATIN.

SATIVUS

Values represent the mean f ments Uv = 211. Treatment

Cisplatin (2 mgkgi Cisplatin (2 mg/kg) + C. sat&u8 (50 mg/kg) C. sativus extract (50 m&kg) Cisplatin (2 mg&gl + N. sativa (50 mg/kg) N. sativa 60 mg/kg) Normal control mice (sterile saline)

S.E.M. of seven mice used per group for three separate experi-

Blood urea nitrogen (mg/lOOml)

P

Day 3

Day 7

24.3 f 1.9 25.7 f 1.2

25.7 2 1.2 22.8 f 0.6

< 0.05

20.0 f 1.6

18.6 + 1.8

< 0.01

18.6 3: 2.3

18.6 2 1.6

< 0.001

22.8 rf: 0.9 21.4 jt 0.8

18.6 f 1.2 21.4 -e 1.9

< 0.001 > 0.05

sativus group, it was increased 194% and with N. sativa extract it was increased only lOoh. At + 10 days, the group of animals receiving cisplatin alone showed a much greater decrease in body weight as compared to animals treated with both cisplatin and C. sat&s (Table 21. This protective effect was not seen in N. sativa-treated animals. Leueocyte counts in mice were predictably altered in the cisplatin-treated group. The leucocyte counts and hemoglobin levels approached normal in both the saffron- and N. sat&a-treated groups receiving cisplatin (Tables 3 and 41. At + 7 days there were significant differences between the blood urea nitrogen levels in mice receiving cisplatin alone and those receiving the combination treatments (Table 61, but there was no significant difference between levels in normal controls and eisplatin-treated mice. Discussion Chemotherapy and ionizing radiation affect non-cancerous cells to a certain extent. Several groups have worked on chemoprotective and radioprotective agents attempting to find a preferential protection of normal tissues during cancer treatment (Yuhas et al., 19801. The most widely used common protective agents are suiphydryl~ontaining compounds like WR-2721 and MPG (Philips, 1980; Milas et al., 19823.In the present study, treatment with a saffron extract significantly prolonged the life span of mice receiving cisplatin. Most of the toxic reactions in cancer therapy with cisplatin are mani-

a2

fested by gastrointestinal epithelium and bone marrow, as evidenced by increased azotemia and gastrointestinal syndromes (Evans et al., 1984; Allan et al., 1984). Most of the chemotherapeutic agents used against malignant disease interfere with cell production by damaging mitotic or cell compartments of the marrow, or by slowing down cell division (Wintrobe, 1976). Hopkins et al. (1976) have reported that the decline of circulating lymphocytes reaches its lowest level on the 5th day of treatment with anticancer drugs. Protection against cyclophosphamide-induced toxicity using MPG has been reported (Bhanumathy et al., 1986). In the present study, blood urea nitrogen levels after cisplatin remained relatively normal indicating no significant renal toxicity. Some protective effects were exerted by both the saffron and N. satiwa extracts considering the lo-day leucocyte counts and blood hemoglobin content. The exact mechanisms by which the saffron and N. sativa extracts exert their protective effects against cisplatin-induced toxicity are not yet known. Acknowledgement

We are thankful to the Indian Council of Medical Research for financial assistance. References Allan, S.G., Cornbleet, M.A., Warrington, P.S., Golland, I.M., Leonard, C.F. and Smytb, J.F. (1984) Dexamethasone and high dose efficacy in controlling cisplatin-induced nausea and vomiting. British Medical Journal 289, 878-879. Allan. S.G., Smyth, J.F., Hay, F.G., Robert, C.F. and Wolf, C.R. (1986) Protective effect of sodium-2-mercaptoethane sulfornate on the gastrointestinal toxicity of cis-diamine-dichloro platinum. Cancer Research 46,3569- 3573. Anonymouse (1982) Cisplatin ten years on. Lancet 1,374- 375. Bannister, S.J.. Sternson, L.A., Repta, A.J. and James, G.W. (1977) Measurement of free circulating cis-diamine-cis-dichloroplatinum (II) in plasma. Clinical Chemistry 23, 2258-2262. Bhanumathy, P. Kumar, S. and Vasudevan, D.M. (1986) Role of 2-MPG against toxicity of cyclophosphamide in normal and tumour bearing mice. Indian Journal of Experimental Biology 24, 767-770. Borch, R.F. and Pleasants, M.E. (1979) Inhibition of cisplatin nephrotoxicity by diethyl-dithio carbamate rescue in a rat model. Proceedings National Academy of Sciences 76, 6611-6614. Comis, R.L. (1980) Cisplatin nephrotoxicity: The effect of dose, schedule and hydrations scheme. In: A.W. Prestoyko, S.T. Crooke and S.K. Carter (Eds.), Cisplutin, Current Status and New Developments, Academic Press Inc., New York, pp. 485-494. Deconti, R.C., Toftness, B.R., Lange, R.C. and Creasey, W.A. (1973) Clinical and pharmacological studies with cis-dichloro-diamine-dichloroplatinum (II). Cancer Research 33, 1310- 1315. Evans, R.G., Wheatley, C.E., Nielsen, J. and Ciborowoski, I. (1984) Modification of the bone marrow toxicity of cis-diamine-dichloroplatinum (II) in mice by diethyldithiocarbamate. Cancer Research 44, 3686 - 3639. Hopkins, H.A. Kovacs, C.J., Looney, W.B., Wakefield, J.A. and Morris, H.P. (1976) Cancer biochemistry. Biophysics 1, 303-306. Howe-Grant, M.E. and Lippard, S.J. (1980) Aqueous platinum (II) chemistry, binding to biological molecules. In: Metal Ions in Biological Systems. Marcel Decker Inc. New York, pp. 63- 125.

a3 Milas, L., Hunter, N., Reid, B.O. and Thomas, H.D. Jr. (19821 Protective effect of S-2-(3-amino propylamino ethyl) phosphorothioic acid against radiation damage of normal tissues and a fibrosarcoma in mice. Cancer Research 42, 1888- 1897. Milas, L., Hunter, N., Ito, H. and Peters, L.J. (19841 Effect of tumour type, size and end point on tumour protection by WR-2721. Internutionul Journal of Radiation Oncology - Biology Physics 10, 41- 48. Phillips, T.L. (19801Rationale for initial clinical trials and future development of radioprotectors. Cancer Clinical T&da 3, 165 - 173. Reishus, J.W. and Martin, D.S. (19611 Cisdichloro-diamineplatinum (II). Acid hydrolysis and isotopic exchange of chloride ligands. Journul of American Chemical Society 83,2457-2462. Riley. CM., Sternson, L.A.. Rept, A.J. and Slyter, S.A. (19821 Reactivity of cis-dichloro-diamineplatinum (II1 towards selected neucleophiles. Polyhedron 1, 201- 202. Rosenberg, B. (19791 Anticancer activity of cisplatin (111 and its relevant chemistry. Cancer Treatment Reports 63, 1433- 1438. Slater, T.F., Ahmed, M. and Ibrahim, S.A. (1977) Studies on the nephrotoxicity of cisplatin (II) and related substances. Journal of Clinical Hematology and Oncology 7, 534-544. Varley, H. (19761 Blood and urine urea. In: H. Varley (Ed.), Practical Clinical Biochemistry. Gulab Vazirani, New Delhi, pp. 156- 167. Wintrobe. M.M. (1974) Pancytopenia, aplastic anemia and “pure red cell” aplasia. In: M.M. Wintrobe, G.R. Lee, D.R. Boggs, T.C. Bithell, J.W. Athens and J. Foerster (Eds.), Clinical Hematology, Lee and Febiger. Philadelphia, pp. 1741- 1776. Yuhas, J.M., Spellman, J.M. and Culo, F. (1980) The role of WR-2721 in radiotherapy and or chemotherapy. Cancer Clinical Trials 3, 211- 216.

Modulatory effects of Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in mice.

In mice, an extract of Crocus sativus stigmas partially prevented the decreases in body weight, hemoglobin levels and leucocyte counts caused by 2 mg/...
545KB Sizes 0 Downloads 0 Views