SCREENING OF SOY LECTIN: AS NEW ERA CANCER HEALING AGENT

SCREENING OF SOY LECTIN: AS NEW ERA CANCER HEALING AGENT

1. T. Tande *¹, M. S. Lade ¹, A. R. Patil ², A. B. Patil ¹ and J. I. D‟souza¹

Tatyasaheb Kore College of Pharmacy ¹, Warananagar, Kolhapur, (MS), India.

D. Y. Patil University ², Centre for Interdisciplinary, Kolhapur 416006. (MS) - 416113, India

ABSTRACT: The lectins are glycoproteins or sugar binding proteins of non-immune origin but are barred from sugar binding antibodies and enzymes. Lectins are isolated and purified from seeds of Glycine max by soxhlet extraction and dialysis. These collected crude lectins were centrifuged till pH is shifted downward to optimal pH for coprecipitation. Filtration of the same carried out on a Buchner funnel with a pad of Hiflo Supercel on whatman paper. Galactose was added as a ligand to the mixture kept at 250C for 10-20 min. It formed matrix coprecipitation which was centrifuged to remove additional particulates. Supernatant was removed and retained the galactose lectin coprecipitate which finally yields lectins, further purified by dialysis. Encapsulation by spray drying using maltodextrin and lactose along with the Eudragit S100 targeted the drug moiety to colon. Purified Lectins have the binding property of carbohydrate moieties on the surface of erythrocytes which agglutinate the erythrocytes, these lectins were evaluated by the agglutination test using ‘A’ positive blood group. These lectins showed anticancer activity against the colorectal type of cancer cell lines HCT- 116; proved as new link for developing anticancer drug specific to colorectal adenomas from Glycine max seeds. Calculated IC₅₀ value by SRB cytotoxic study which was found 12 compared with capecitabine as standard anticancer drug which was

Soybean (Glycine max),

Lectins, Colon Cancer

INTRODUCTION: Lectins and History: Term “lectin” is from the Greek word “legere” was coined by William Boyd in 1954, “legere” means “to select” or “to unite” ¹. The lectins are described as sugar binding proteins or glycoproteins of non-immune origin excluded from sugar binding antibodies and enzymes ². Lectins are widely distributed from bacteria to humans, which have as a minimum one non-catalytic area that shows reversible binding to particular monosaccharides or oligosaccharides. Lectins are easily detected by agglutination of erythrocytes. Lectins are classifieds on the basis of their overall structure and biochemistry of the subunits viz., merolectins, hololectins, chimerolectins and superlectins

Soybean and chemistry of soybean lectins:

Soybean (Glycine max; Family: Fabaceae) plant is member of legume family native to East Asia. Soybean lectin is a protein, which have ability to bind with the carbohydrate so called as the glycoprotein. They have high binding protein specificity to fatal non-reducing N-acetyl-D-galactosamine but less to D-galactose. They have important role in cell to cell communication ^{5, 7}.

Soybean also decreases the chances of breast and prostate cancer. They reduce the protein digestion due to the presence of the protein inhibitor^{5, 7}.

Objectives:

• Generation of crude lectin via extraction
• Characterization of soybean lectin
• Generation of pure lectins
• Anti-cancer activity of soybean lectin via In-Vitro studies

MATERIAL AND METHODS:

Soxhlet Extraction:

Soybean seeds were finely grounded and was weighed accurately about 100 g, then wrapped in filter paper and placed in soxhlet apparatus for extraction. Different extraction solvents were used separately for isolation of lectins as acetone, 0.015 M phosphate buffer of pH 7.2 in 0.15 M NaCl and methanol (500 ml each). Extraction was carried out for 1-1.30 hour with continuous stirring.

 Co-Precipitation Procedure:

Collected lectins processed for centrifugation which was carried out to remove bulk particles. Coprecipitation was formed by shifting pH downward to optimal pH and the solution is kept overnight in the cold room. Remaining particulates removed by a combination of centrifugation; filtration was carried out on a Buchner funnel with a pad of hiflo supercel on whatman no. 1 paper. Obtained clear solution was adjusted to optimal pH. Galactose ligand added to the mixture and kept for 10-20 min. at 25⁰C to form matrix coprecipitation and was centrifuged to remove additional particulates. Supernatant was removed and retained the galactose lectin coprecipitate, then dissolved in buffer of pH 8 and trapped on anion exchange resin in Cl^- form. Received lectins were evaluated and processed for dialysis for purification.

Evaluation of Raw Lectins

Agglutination Test ²¹: Isolated and collected lectins were processed for agglutination test. Lectins were placed in different tubes and mixed with human blood of A^+ve blood group.

Hemagglutination Assay ²¹:

Lectins have the property to agglutinate the erythrocytes. Soybean lectin forms a mesh like structure in between the blood erythrocytes which do not let the blood to be clumped. Assay was performed in two groups as positive and negative control.

Dialysis ¹⁷:

It is common operation in biochemistry and protein biology which assist to separate dissolved molecule by passing through the semi-permeable membrane as per their molecular dimensions.

Spray Dry Method ^{19, 20}:

The spray-drying process of lectin done using the various excipients was undertaken in a laboratory scale spray dryer. The feed solution was pneumatically atomized into a vertical, concurrent drying chamber using a two-fluid nozzle at a constant flow rate (5ml. min^‑1 to 20ml. min^-1). The outlet temperature was adjusted from 110^OC to 120^OC by varying the air inlet temperature. The dried powder was collected in a single cyclone separator.

TABLE 1: RATIO OF LECTIN AND EXCIPIENTS TAKEN FOR SPRAY DRYING

Hemagglutination Assay for Identification of Optimized Batch:

Evaluation test were carried out to evaluate the optimized batch for spray dried lectins. Soybean lectin showed hemagglutination activity with blood group type of A^+ve

Screening of Anticancer Activity; In-Vitro Study:

Cell line and Drugs:

The human colon cancer cell line (HCT 116) purchased from national centre for cell science,

Pune (N.C.C.S.) and used for in-vitro anticancer study.

Chemicals used were; 1, 2-dimethyl hydrazine- Purchased from Sigma Aldrich CAS No.-406-16-2, Capecitabine (Gift sample from Cadila pharmaceuticals Ltd. Ahmadabad)

 SRB Assay ²²:

Sample Preparation:

Stock solution of lectin powder of 1 mM in 0.25% DMSO was prepared and further dilution was done in 10, 50 and 100µM with phosphate buffer saline (PBS).

Stock solution of Capecitabine of 1 mM in distilled water was prepared and further dilution was done in 10, 50 and 100µM with phosphate buffer saline (PBS).

Experimental Procedure:

• Counted the cells and adjusted the concentration of the cell suspension

• Added 100 μl of a cell suspension to each well in 96 well microplate using serial dilutions. Made a well of only media to measure the background

• Incubated for 24 hours in a CO₂ incubator with 5% CO₂

• If media change is necessary, remove media and add 100 μl of new media to each well including wells for a background measurement

• Added 10 μl of media containing different concentrations of the test substances to each well

• Incubated for set periods of (24 hrs) in a CO₂ incubator

• Added 10 μl of SRB solution to each well in a 96 well microplate

• Placed in a CO₂ incubator 5% CO₂ for 1-4 hours to react

• Measured the absorbance at 450 nm with a microplate reader

Calculation:

Enter the absorbance reading from each well in the equation below to calculate the cell survival rate.

Survival rate (%) = (A _sample – A _b / A _c –A _b) × 100%

RESULTS AND DISCUSSION:

Agglutination Test:

Test carried out for agglutination was found positive for lectins in PBS which was used as solvent for soxhlet extraction.

 Hemagglutination Assay:

For this assay human blood sample of A^+ve blood group was used. Tests were compared with positive and negative control assay. PBS had shown the positive results for lectins.

 Spray Dry Method:

 TABLE 2: DRUG-EXCIPIENTS RATIO STUDY FOR SPRAY DRYING

*Selected batch for activity as per agglutination studies result

 Screening of Anticancer Activity; Comparative In-Vitro Studies (SRB Assay):

Cytotoxic activity of Methanolic extract of lectin (L. P. A. test) and Capecitabine were investigated against SRB assay and cytotoxicity was reported in terms of lethality concentration (LC50). The LC50 value of batch A, batch B, batch C, batch D and Capecitabine were 80 μg/ml, 75 μg/ml, 15 μg/ml, 50 μg/ml and 9 μg/ml respectively by SRB assay. Show in Table 3, 4, 5 and 6 and Fig. 1, 2, 3 and 4 respectively.

 TABLE 3: SRB ASSAY FOR BATCH A

All values expressed in Mean ± S.E.M. (n=3)

 TABLE 4: SRB ASSAY FOR BATCH B

All values expressed in Mean ± S.E.M. (n=3)

 TABLE 5: SRB ASSAY FOR BATCH C

All values expressed in Mean ± S.E.M. (n=3)

 TABLE 6: SRB ASSAY FOR BATCH D

All values expressed in Mean ± S.E.M. (n=3)

SUMMARY AND CONCLUSION: The present study was based on finding of anti-cancer activity of lectins obtained from the soya seeds. These days many synthetic agent used for curing many disorders like cancer and other diseases but they are available with many side effects. Consequently it was decided to generate such an agent with least or null side effects and corresponding to natural flora of Gastrointestinal tract.

Finally it was concluded that;

• Crude lectins were obtained from the PBS

• Lectins can be purified by the dialysis method

• Encapsulation with the excipients like starch and maltodextrin along with Eudragit S100 can be used for colon targeting of lectin moiety

• New anti- Cancer activity was screened via In-Vitro test successfully

 REFERENCES:

1. Islam B and Khan AU: Lectins: to combat infections, protein purification. Rizwan Ahmad (editor). InTech 2012; 167- 188.
2. Jimbo M, Satoh S, Hasegawa H, Miyake H, Yoshida T, Maruyama T and Kamiya H: The difference of lectin recovery by sugar-bound resin, affinity chromatography. Dr. Sameh Magdeldin (editor). In Tech 2012; 75-88.
3. Basu D and Appukuttan PS: Plant lectins specific for N-acetyl-β-D-galactosamine. Bioscience Vol. 5, December 1983; Supplement- 1: 131-135.
4. Ambrosi M, Cameron NR and Davis BG: Lectins: tools for the molecular understanding of the glycocode. The Royal Society of Chemistry. Org. Biomol. Chem. 2005; 3: 1593-1608.

5. Bhol CS: Isolation and characterization of soybean (Glycine max) lectin. Department of Life Science National Institute of Technology. Rourkela- 769008, 1- 32.
6. Fülöp N, Marchase RB and Chatham JC: Role of protein O-linked N-acetyl-glucosamine in mediating cell function and survival in the cardiovascular system. European Society of Cardiology 2006; 73: 288- 297.
7. Dixit AK, Antony JIX, Sharma NK and Tiwari RK: Soybean constituents and their functional benefits, challenge and scope of natural products in medicinal chemistry 2011; 367- 383.
8. Gupta D, Dhiman K, Harisha CR and Shukla VJ: pharmacognostical and physico-chemical evaluation of soya seed granules. International Journal of Pharmaceutical & Biological Archives 2013; 4(1), 96- 99.
9. Bernstein C: Inflammatory Bowel Disease: a global perspective, World Gastroenterology Organization Global Guidelines. 2009; 1-24.
10. Lewandowicz G, Harding B, Harkness W, Hayward R, Thomas DG and Darling L: Chemosensitivity in childhood brain tumors in-vitro: evidence of differential sensitivity to Lomustine (CCNU) and Vincristine. Cancer 1955-1964; 36.
11. Tanaka T: Colorectal Carcinogenesis: Review of human and experimental animal studies, journal of carcinogenesis 2009; 8, 1-19.
12. Tortora G and Grabowski S: Principles of Anatomy & Physiology. 10^th John Wiley & Sons Inc. New Jersey, 2003; 853-855, 891-893.
13. Boveri T: Tumour Suppressor Genes & factors affecting 1914; Available on: http://beta.garlandscience.com/res/pdf/9780815340782_ch07.pdf.
14. Levi E, Misra S, Du J, Patel B and Majumdar P: Combination of aging and dimethylhydrazine treatment causes an increase in cancer- stem cell population of rat colonic crypts. Biochemical and Biophysical Research Communications 2009; 385, 430-433.
15. Byrne L: The details of colorectal cancer (2008); Available on: http://www.cwru.edu/med/epidbio/mphp439/Colorectal_Cancer.pdf.
16. Rangari VD: General methods of extraction, isolation and purification, pharmacognosy and Phytochemisry. Career Publications Vol. I: 97- 98.
17. Wu CW, Lovrien R and Matulis D: Lectin coprecipitative isolation from crudes by little rock orange ligand. Analytical Biochemistry 1998; 33- 39, 257.
18. Sattayasai N: Protein purification. Chemical Biology. Deniz Ekinci (Ed.) 2012; 3-18
19. Albertini B, Passerini N, Pattarino F, Rodriguez L: New spray-congealing atomizer for the microencapsulation of highly concentrated solid and liquid substances. European Journal of Pharmaceutical and Biopharmaceutical science 2008; 69: 348–357.
20. Woo MW, Huang, LX, Mujumdar AS, Daud WRW: Computational fluid dynamics modelling of spray dryers, Spray Drying Technologyy. 2010; Vol- 1: 1-36.
21. Power L: Dietary lectins: blood types & food allergies, townsend letter for doctors (1991), 1-17.
22. Voigt W: Sulforhodamine B assay and Chemosensitivity. Methods in Molecular Medicine. Vol. 110, Chemosensitivity: Vol. 1: In Vitro Assays, 39-48.
23. Brad K and Chen C: Physicochemical properties of diosmetin and lecithin complex. Tropical Journal of Pharmaceutical Research 2013; 12 (4): 453-456.
24. Chanda S and Nagani K: In vitro and in vivo methods for anticancer activity evaluation and some Indian medicinal plants possessing anticancer properties: an overview. Journal of Pharmacognosy and Phytochemistry. 2013; Vol. 2: 140- 152.
25. Sreelatha D, Brahma CK: Colon targeted drug delivery– a review on primary and novel approaches, Journal of Global Trends in Pharmaceutical Sciences. July-September 2013; Volume 4: Issue 3: 1174-1183.
26. More SK and Wagh MP: Review on spray drying technology. International Journal of Pharmaceutical, Chemical and Biological Sciences 2014; 4(2): 219-225.
27. Bhokre C, Ghatge PU, Machewad G and Rodge A: Studies on preparation of buns fortified with germinated horse gram flour. Open Access Scientific Reports, 2012; Vol. 2: Issue- 4: 1-2.
28. Muthu R: Synergistic and individual effects of umbelliferone with 5-flurouracil on the status of lipid peroxidation and antioxidant defense against 1, 2-dimethylhydrazine induced rat colon carcinogenesis. Journal of Biomedicine & Preventive Nutrition 2012; 10: 1-9.
29. WishartD: Maltodextrine 2013; Available on: http://www.drugbank.ca/ molecules/3668
    

    ---

    How to cite this article:

    Tande AT, Lade MS, Patil AR, Patil AB and D’souza JI: Screening of Soy Lectin: As New Era Cancer Healing Agent. Int J Pharm Sci Res 2016; 7(5): 2147-51.doi: 10.13040/IJPSR.0975-8232.7(5).2147-51.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---