1. INTRODUCTION:

Protease inhibitors are a group of small proteins from non-immune origin that share the property of binding to the target proteases¹. Many plant protease inhibitors have attracted the attention due to their effects on the proliferation and differentiation of the animal cells, including cancer cells. The in vitro and in vivo anti tumor effects of the protease inhibitors are apparently associated with their modulate growth, differentiation and proliferation². Toxicity of the protease inhibitors must be considered as the major step to use as medical tools, mainly because of the anti-nutritional factors. They are known to interfere in the nutrition absorption and negative absorption on digestion has been described from plant derived protease inhibitors³.

Many plant derived protease inhibitors have been proved to posses anti cancer activity⁴. Before testing the anti cancer activity of the soy bean (SB), we studied the acute toxicity of SBPI using OECD guidelines 423 and were assayed for sub chronic toxicity (daily dosing for 14 days).

Many researches proved that diets containing high amounts of soy bean products are associated with low cancer incidence and mortality rates particularly against breast cancer. Breast cancer is one major concern to the world majority in the western world⁵. Japan has the highest consumption of soy bean (SB) products suggested to lead the extremely low cancer mortality rates^5,6. Many studies have proved that Bowmann Birk Inhibitors (Protease inhibitors) (BBI`s) in the soyabean leads to the anti cancer mortality. Along with the BBI`s, isoflavones, β-sitosterol, phytic acids are also held responsible for the anti-tumor activity⁷.

Recent scientific studies proves that many different kinds of PI`s in the pure form suppress carcinogensis^8,. The partially purified protease inhibitor form SB characterized ability to inhibit trypsin and chymotrypsin and has been reviewed elsewhere^{9, 10}. SBPI has been studied in the purified form, but partially purified form has not been studied for the toxicity profile and the anti cancer activity. Some of the human cancer clinical trials are also registered over the purified forms of SB.

There are economical and purification concerns over the complete purification of the protein. So, in the present study we are herewith to evaluate the partially purified protein of SBPI to screen the anti cancer and toxicity studies. Hence, there is need to evaluate the economically commercially feasible as well as environmental friendly preparation of the SBPI in order to meet the growing demand in the anti cancer drug sector. Partially purification technique uses the economical, efficient and eco-friendly procedures they may be used as the regular homicidal proteins. The major advantage of the partial purification is their important role in protecting the bodily system and also easy preparation technique.

Here we present short term assay in order to the effects SBPI for evaluating the toxicity and anti cancer activities. Earlier studies have reported the effect of purified protein activity but no activity reported on the toxicity profiles and anti cancer activities on the partially purified SBPI against MCF-7 and Vero cell lines in similar doses and comparative studies were also performed between animal toxicities.

2. MATERIALS AND METHODS:

RPMI-1640 medium, trypsin/ EDTA solution, fetal bovine serum, glutamate-penicillin-streptomycin solution were purchased from sigma chemical Co., St Louis USA. All other analytical solvents, de-ionized water and sodium citrate were procured from Fischer Scientific, India.

2.1 Experimental animals:

The wistar rats were procured from the Venketeshwara Enterprises, Bangalore and placed in individual cages with ad libitum water and rodent chow pellet diet. The animals remained one week for acclimatization for adjustment to 12h light/12h darkness, at 22°C and a relative humidity of 30 %. The animals were sacrificed at the end of the experiment by decapitation. The experimental protocol was approved by the Institutional Animal Ethics Committee of KLEU`s College of Pharmacy, Belagavi which has registered with CPCSEA, Govt. of India (registration no 221/CPCSEA) with resolution no (KLECOP/IAEC/Res 18-19/05/2014)

2.2 Plant collection:

The plant material was collected from the local fields of Belagavi, Karnataka. The Plant has been identified and authenticated by the Scientist C, ICMR (RMRC), Belagavi as Glycine max (L.) Merr. (Fabaceae) voucher specimen (RMRC 1264) was preserved in the same department.

2.3 Partial purification of the protease inhibitor:

The 100 grams of the seeds were made to coarse powder and homogenized in 1000 ml of the 0.1M Phosphate buffer (pH 7.0). The solution was then filtered through the four layered cheese cloth. The filtered solution was then centrifuged at 10,000rpm/15mins/4°C (Kubato cooling centrifuge). The supernatant was collected and the precipitate was discarded. The supernatants were further fractionated by ammonium sulfate (30, 60, and 90%). The 90% fraction was collected by adding minimum quantity of the phosphate buffer. The collected suspension was dialyzed for 24 hours at 4⁰C in 0.1 M phosphate buffer (pH 7.0). The dialyzed suspension was then lyophilized and stored in -80°C until further use. The proteins were assayed by Lowry et al method using bovine serum albumin as standard. The purification was performed regularly for until the end of the experiment¹¹.

2.4 Acute Toxicity studies:

Acute toxicity studies were performed according to OECD guide lines 423. The five rats were fasted for six hours and administered with single oral dosage of 2000 mg/kg and observed for mortality for 24 hours.

2.5 Sub chronic oral administration of SBPI:

The six rats (n=3) were fasted for 12 hours before oral administering with 200mg/kg of SBPI to treatment group and other group received the water ad libitum for 15 days. Food intake and water intake was measured on day 1, 7, 15^th day prior to the treatment. Blood was collected by retro orbital plexus in EDTA coated tubes. On the final day, animals were sacrificed by decapitation method and body organs like liver, kidney, heart, spleen and pancreas were separated and weighed. After 15 days, the complete blood count was estimated and compared with the control. Liver function was analyzed by Asparate aminotransferase (AST), SGOT, SGPT, total protein. Other parameters like glucose, insulin, and creatinine were also measured using the commercial kits.

2.6 In vitro cell viability and cytotoxicity assays:

2.6.1 Cell culture:

The study was carried out in MCF- 7 breast cancer cell lines, were obtained from National center for cell science (NCCS), Pune, India and cultured as monolayer in RPMI-1640 medium supplementated with 10% fetal bovine serum (FBS), penicillin and streptomycin.

Vero cell line culturing technique Procedure:

Vero cells are derived from the kidney of an African green monkey were obtained from National center for cell science (NCCS), Pune, India and cultured as monolayer in Dulbecco’s modification of Eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), filter sterilized.

2.6.2 Evaluation of cytotoxicity assay:

The growth inhibitory activity of cells was determined by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay based on the detection of mitochondrial dehydorgenase activity in the living cells. (Kartikeyan et al 2013). Cells were grown (1x10⁴ cells/well) in a 96 well plate for 24 h with 80% confluence. The old medium was replaced with fresh medium containing serially diluted PPI and standard and the cells were further incubated for 24 hours removing and the cells were further incubated for 24 h removing the culture medium after 24 h, 10µL of MTT solution (5mg/L in PBS) was added and again allowed for 4 hr incubation at 37 ⁰C towards color reaction development. An equal volume of DMSO was added to stop the reaction and to solubilize the blue crystals. Absorbances were measured using microplate reader at a wave length of 570 nm.

2.7 Statistical analysis:

All the parameters were expressed as Mean ±SD and ANOVA was carried out followed by post Dunnett`s multiple comparison t- test using Graph pad Prism 5 statistical software. The values of anti cancer study are given as Mean ±SD for triplicate samples in each group. Difference between the groups were considered significant at p<0.05.

3. RESULTS AND DISCUSSIONS:

3.1Acute oral toxicity studies:

Acute oral toxicity studies were performed according to the OECD guidelines 423 and found no mortality even after administering 2000mg/kg single oral dose. 1/10^th dose of the dose (200 mg/kg/BW) of SBPI was chosen for screening sub acute toxicity studies and control group received water.

3.2 SBPI response after 15 days of drug administration:

200 mg/kg SBPI dose was administered via intragastric route every day until 15 days. Significant decrease in food consumption was observed from the first week of administration until 15^th day respect to the control group. However, on the last week food consumption was as similar to the control group, may be due to the compensatory mechanisms where the treated animals overcame the negative effects of the protease inhibitors administration. Rats body weights also showed significant changes (p<0.05) between the two groups. There was transient decrease in bodyweights in the first weeks (%) however; at the end of the study a recovery of weight was observed resulting in reduction in bodyweight of 10 % respect to the control group. It is well known that PPI interfere with the nutrition absorption, resulting in animal nutritional status. Our results show that SBPI administration causes anti-nutritional effects at the beginning of the experiment with a final recovery, which resulted in reduction in body weight gain.

3.3 SBPI administration to the change in the blood markers:

The effect of SBPI on organs and blood markers is shown in table-1 except the granulocytes were significantly higher than the control. We observed no significant difference in spleen, heart, liver, kidney, stomach, thymus, pancreas in compared to the control group. Results are illustrated in table-2.

Table: 1 Complete Blood Count after 15 days of administration of SBPI oral administration.

S. No	Blood Parameter	Control	SBPI
1	WBC(10³/µL)	5.44±0.20	5.84±0.45
2	Lymphocytes (10³/µL)	4.74±0.24	4.61±0.12
3	Granulocytes (10³/µL)	0.46±0.08	0.50±0.041*
4	RBC (10⁶/µL)	6.72±0.17	6.32±0.11
5	Platelets (10³/µL)	760.77±29.82	812.13±41.22
6	Hb (g/dL)	12.33±0.22	12.23±0.48

Statistically significant difference (t test,p<0.05)

Table: 2 Organ weights after SBPI sub chronic oral administration

S.No	Blood Parameter	Control	SBPI
1	Heart (g)	1.69±0.07	1.67±0.02
2	Kidneys (g)	2.81±0.41	2.88±0.45
3	Liver (g)	11.88±0.17	12.13±0.49
4	Spleen (g)	0.85±0.04	0.87±0.03
5	Pancreas (g)	1.58±0.14	1.60±0.03

No statistically significant difference was founded (p>0.05)

Table: 3 Blood markers after the sub chronic oral administration of SBPI

S. No	Blood Parameter	Control	SBPI
1	Serum insulin levels(µIU/ml)	3.38±0.35	3.416667±0.40
2	Serum SGOT(U/L)	118.83±3.18	124.3333±8.57
3	Serum SGPT(U/L)	96.5±7.86	98±7.745967
4	Serum ALP(U/L)	180.33±3.77	187.66±13.80
5	Glucose	79.16±9.62	75.83±10.88
6	Serum total protein (g/dL)	7.74±0.52	7.60±0.29
7	Serum cholesterol (mg/dL)	67.9±4.85	66.96±12.08
8	Triglycerides (mg/dL)	60.73±3.51	64.38±4.65
9	Serum creatinine(mg/dL)	0.72±0.12	0.77±0.07

No statistically significant difference was found (p>0.05)

Different blood markers were determined on the last day in order to study hepatotoxicity (SGOT, SGPT), renal injury (creatinine), and nutritional status (albumin, total protein, creatinine, and glucose). Results are illustrated in table-3. No differences between groups were found suggesting that the oral SBPI administration exhibit no toxicity at the end of the treatment. No significant differences between groups were found, suggesting no adverse effects after the sub acute toxicity studies.

3.4 Dose dependent cytotoxicity/cell viability of SBPI in MCF-7 and vero cell lines:

The cell viability assay is one of the important methods for toxicological analysis which explains the cellular response to the toxic materials and it can provide information on cell death, survival and metabolic activities¹².

Recently, Magee PJ et al reported that PI purified from Cicer arietinum showed cytotoxicity in dose dependent manner in MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells cell lines¹³.

Earlier studies have demonstrated that Bowman birk inhibitors purified from soya bean using extract induced dose dependent cytotoxic effect on lung tumors ¹⁴, esophageal neoplasms¹⁵ and pancreatic neoplasms cell lines¹⁶.

In the present work, we selected breast cancer cells MCF-7 for our study because they are most frequent cell lines chosen for the breast cancer studies and normal kidney cells (Vero cell lines) for evaluating the cyto-protective activity. Vero cells are well suited for mechanistic and toxicological studies¹⁷.

The above cell lines were also chosen to compare the cytotoxicity of PI in cancer and normal cells that differentially respond to PI. To examine the effect the of both cells which are treated with various concentrations (0-120µg/ml) of SBPI for 24 h and the results suggest that SBPI were able to reduce the cell viability of MCF-7 cells in a dose dependent manner.

The most apparent and noticeable affect following treatment of cells of SBPI shows MTT reduction capability of SBPI in MCF-7 cell lines with significantly 80 and 120 µg/ml.

Further, we observed the partially purified SBPI treatment at concentrations (0-120µg/ml) has not impacted the percentage viability in normal human kidney cell lines (Vero) cells. In contrast, they are effective against the MCF-7 cells. The cell viability assays confirmed that no significant was found toxicity in normal vero cells.

In agreement with our results other research groups have reported that cell viability was significant reduced the IC₅₀ of PI at 75 µg/ml against MCF-7 and 120 µg/ml in normal cells.

4. CONCLUSIONS:

Here we report that sub chronic administration of SBPI proved the anti nutritional effects in rats resulting in transient decrease of food intake and body weight in the first weeks. The final results were observed as a reduction in body weight gain in respect to the control group. Blood markers suggest no toxic effects and no alternations in the evaluated groups. Taking together, our results showed that SBPI provoke a reduction in body weight gain with no other remaining effects, suggesting compensatory mechanisms and good tolerability. SBPI also proved a good anti cancer agent against the MCF-7 cell as well as cyto protective against the normal cells Vero cells.

5. CONFLICT OF INTEREST:

Authors declare no conflict of interest

6. REFERENCES:

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13. Pamela J. Magee, Richard Owusu-Apenten and Mark J et al . chickpea (Cicer arietinum) and Other Plant-Derived Protease Inhibitor Concentrates Inhibit Breast and Prostate Cancer Cell Proliferation In Vitro. Nutrition and Cancer 64(5); 2012:741-8.

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Received on 27.06.2016 Modified on 02.07.2016

Research J. Pharm. and Tech. 2016; 9(7):925-928.

DOI: 10.5958/0974-360X.2016.00177.3

Recently, Magee PJ et al reported that PI purified from Cicer arietinum showed cytotoxicity in dose dependent manner in MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells cell lines13.

1. Antao CM and Malcata FX. Plant serine proteases: biochemical, physiological and molecular features. Plant Physiology and Biochemistry. 43(7); 2005: 637-650.

13. Pamela J. Magee, Richard Owusu-Apenten and Mark J et al . chickpea (Cicer arietinum) and Other Plant-Derived Protease Inhibitor Concentrates Inhibit Breast and Prostate Cancer Cell Proliferation In Vitro. Nutrition and Cancer 64(5); 2012:741-8.

Recently, Magee PJ et al reported that PI purified from Cicer arietinum showed cytotoxicity in dose dependent manner in MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells cell lines¹³.