(Amala Research Bulletin (1992): (12), 49)

Immunomodulatory Activity of Septilin

Praveen Kumar, V., Kuttan, G. and Kuttan, R., Amala Cancer Research Centre, Thrissur, Kerala, India.

ABSTRACT Administration of Septilin was done in untreated animals as well as in the tumour bearing animals. The preliminary analysis indicated that Septilin was found to be non-toxic and concentrations more than 100 mg in hot water on alternate days for one month did not produce any toxic symptoms. Haematological analysis showed that Septilin increased the total counts of leukocytes as well as produced a preferential increase in the Polymorphonuclear cells. This indicated that administration of Septilin not only stimulated the leukocyte production but also increased their differentiation. Septilin was not found to be directly cytotoxic to tumour cells when added as either alcoholic or aqueous extracts. Administration of Septilin (100 mg/animal on alternate days for one month) increased the survival of animals bearing Ehrlich ascites tumour. The ILS calculated was 70.6%. Thus our preliminary evidence indicated its usefulness as an immunomodulator in cancer therapy. INTRODUCTION Septilin, an Ayurvedic preparation prepared by The Himalaya Drug Co., Bombay, contains herbomineral Principles which have antibacterial, anti-inflammatory and antiexudative properties. It is extensively used in the treatment of several acute/chronic infection. (1) It has been reported that it increases the phagocytic coefficient (2) which corresponds with clinical improvement in chronic infections resistant to the commonly used broad spectrum antibiotics. In the present study we examined whether Septilin is an immunomodulator that has the capability to cause tumour regression in tumour bearing animals. MATERIAL AND METHODS Extraction: Six grams of Septilin powder was supplied by The Himalaya Drug Co., Bombay, was boiled in 600 ml of distilled water for 30 min. It was centrifuged and the filtrate evaporated to dryness on waterbath and made upto 45 ml. 075 ml of the extract was used for oral administration which contained extract from 100 mg of Septilin. Haematological studies: Two sets of Swiss albino animals, six mice in each set, were used. One set acted as controls and to the other 100 mg of hot water extract of Septilin was given orally on alternate days for one month. The blood was collected by bleeding the tail vein on every three days. Total and differential counts of leukocytes and haemoglobin levels were checked both before and after treatment using standard procedures. In vitro cytotoxicity assessment Ehrlich ascites and Sarcoma-180 cells (106) grown as ascits cells in mice were incubated at 37C in presence of various concentrations of the aqueous and ethanolic extracts of Septilin for 3 hrs. After incubation the percentage of live cells was determined using trypan blue exclusion method. (3) Tumour reduction studies In-bred strains of swiss albino mice were used for tumour reduction experiments. Transplantable Ehrlich ascites tumour cells (1 106 cells) were injected intraperitoneally into the animals [6

animals each]. After 24 hrs. 100 mg of hot water extract of Septilin was given per animal orally from day one and then continued on alternate days for one month. Mortality of animals dying of tumour were noted and average increase in life span was calculated by the formula (4). Percentage increase in life span = TC C
100

[T average life span of treated mice, C average life span of control]. In another experiment, five sets of inbred swiss albino mice (three in each group) were given transplantable Ehrlich ascites tumour cells (106) intraperitioneally. Twenty four hrs. after transplantation, 100 mg of Septilin was given orally on alternate days for one month starting form the day one. The controls were left untreated. On every 5th day each set was sacrificed and the peritoneal cavity was washed thoroughly and the number of live cells were counted. RESULTS Effect of Septilin on the haematological parameters: When the animals were treated with Septilin (100 mg/animal) on alternate days for one month there was a significant increase in the total count of leucocytes (p > 0.001) and reached a peak on 9th days (Fig.1). Similarly the percentage of polymorphonuclear cells were also found to increase in the drug treated animals and gave a peak on 9th day (Fig.2). There was no significant change in the haemoglobin levels or the body weight of Septilin treated animals compared to controls under the conditions of the experiment.

Fig. 1 Effect of Septilin on WBC counts in albino mice

14 12

Total Counts (10 3)

10 8 6 4 2 0 0 3 6 9 12 15 18 21

No. of days Drug Treated Control

Effect of Septilin in the reduction of ascites tumour Table 1 shows the effect of administration of Septilin orally 24 hrs. after transplantation of tumours. The untreated control animals survived only for 17 days. But the Septilin treated group survived 29 days. The percentage increase in life span of Ehrlich ascites tumour bearing animals calculated were 70%.
Table 1: Effect of Septilin on tumour Bearing Animals No. of days % ILS survived Control 17.33 3.93 Treated 28.50 2.07 70.58

Fig.2: Effect of Septilin on differential count in albino mice

The results of tumour reduction by counting the number of cells on every 5th day is given in Fig.3. A slight decrease in number of cells can be seen on 5th, 10th, 15th and 20th day. Controls did not survive more than 25 days. Septilin treatment increased the life span of the tumour bearing animals.

Lymphocyte 100 80

60

40 20

% of cells

Fig.3: Effect of Septilin on the tumour cell number in peritoneal cavity

320 280

0 3 40 PMNS 6 9 12 15 18 21

No. of Cells (106)

240 200 160 120 80 40 0 0 10 20 30

30

20

10

0 0 3 6 9 12 15 18 21

No. of Days Drug Treated Control

No. of days Drug Treated Control

In short term in vitro cytotoxic assay using trypan blue exclusion method, gave no significant cytotoxicity to both Ehrlich ascites and S - 180 tumour cells, when added either as alcoholic or aqueous extracts (Table 2).
Table 2: In vitro cytotoxicity of Septilin to Ehrlich ascites tumour cells Extracts Con. Of drug (mg) % of cell death 20 47 Cold water 10 43 5 34 20 57 Hot water 10 39 5 20 20 46 Methanolic 10 27 5

DISCUSSION It has been reported that Septilin is non-toxic and free from side effects (3). In our study it gave no toxic symptoms even at concentrations upto 500 mg for one month. It was well tolerated even after prolonged use. Like all immunomodulants, Septilin was also found to increase the total count of leukocytes significantly and the percentage of polymorphonuclear cells. This suggest the immunopotentiating activity of Septilin.

In the tumour reduction studies, it did not show cyto toxic activity, directly to the tumour cells in the short term in vitro assay. But the treatment of mice with Septilin increased the percentage increase in life span of tumour bearing animals of about 70%. Hence we suggest that this increase may be due to immunopotentiating activity of Septilin and thus our preliminary experiments suggests the usefulness of Septilin as an immunomodulator in the cancer treatment. REFERENCES 1. D.G. Ross (1984) The anti-infective and antibacterial efficacy of Septilin; Probe, 23, 84-86. 2. M.R. Bhat (1983) The phagocytic activity of Septilin in chronic recurrent injections: Probe, 22, 100-103. 3. Kuttan R., Bhanumathy P., Nirmala K. and George M.C. (1985) Potential anti-cancer activity of turmeric (Curcuma longa). Cancer letters, 29, 197-202. 4. Unnikrishnan M.C. and R. Kuttan (1990) Tumour reducing and anti-carcianogenic activity of selected spices: Cancer letters 51, 85-89.