INTRODUCTION:

Role of proteases in the early stage tumor growth and progression followed by invasion and metastasis has been studied and the need for protease inhibitors has been highlighted (Jennifer et. al., 2000). Protease inhibitors like trypsin and chymotrypsin inhibitors are known to inhibit the enzymatic activity leading to reduced physiological event progression. These inhibitors are mostly proteinaceous and are very specific in function. Protease inhibitors are broadly studied in microorganisms, plants and animals (Hamao 1976, Caroline et. al 2013, Leiner et. al., 1980).

Plants are known to contain abundance of protease inhibitors which can be easily isolated and purified. These inhibitors can also be commercialized for use in therapeutic applications. Two trypsin inhibitors were isolated and purified from the leaves of tomato using salt precipitation and Sephadex based gel filtration chromatography (Gregory et. al., 1982). These inhibitors were also found to inhibit chymotrypsin and subtilisin up to an extent. The molecular weights of these inhibitors were in the range of 41,000 Da and 23,000 Da. Pea is known to contain protease inhibitors which was purified on a Sephadex platform with a total activity of ~50% and molecular weight in the range of 7000 Da. These inhibitors are known to act as plant defense against insects (Amrit et. al., 2016). A protease inhibitor with molecular weight of 39,000 Da was isolated from tobacco separated on a Sephadex G-75 platform (Tsung-Min 1984).

A review on Senna alata discusses about the medicinal properties of this plant which belongs to shrub family and found in intertropical areas. Being native of central America, this plant also shows its presence in Caribbean area. Few of the medicinal applications to which Senna alata is used include digestive related, dermatologic, antiinfectious, antidiabetic and some of the inflammation and pain related conditions (Hennebelle et. al., 2009). Extracts from Senna alata has demonstrated anti-lipogenic effects (Jarinyaporn et. al., 2016). In this study, we have successfully isolated and purified two protease inhibitors showing trypsin inhibitor activity separated using Sephadex G-10 followed by Sephadex G-50 based gel filtration chromatography. Reverse phase high performance liquid chromatography was use for further purification of these inhibitors.

MATERIALS AND METHODS:

Materials:Senna alata seeds were collected from surroundings of Puthige panchayath of Kasaragod district of Kerala State.

Chemicals: Bovine Serum Albumin, trypsin, BAPNA, α-amylase, Starch, Sephadex G-10 and Sephadex G-50 were purchased from Sigma chemical laboratory, USA. All the other chemicals and reagents used were of technical grade.

Methods:

Extraction of protease inhibitor and purification

Crude protease inhibitor extract was prepared using sodium phosphate buffer pH 7.0 by stirring over a magnetic stirrer for 1.5 hr at 4^o C from acetone powder of soaked seeds of Senna alata which was prepared by blending with chilled acetone for 5 – 6 minutes as described by Chandrashekharaiah (2013). It was then centrifuged at 10000 rpm for 15 minutes. The supernatant obtained was used as inhibitor extract. Solid powdered ammonium sulphate was added slowly to inhibitor extract with stirring over magnetic stirrer at 4^oC for 30 minutes to obtain 0 - 85% saturation. After the addition of all the salt stirring was continued for 30 minutes and allowed to stand at 4^oC for 3 hours followed by centrifugation at 10000 rpm for 30 minutes. The pellet obtained was dissolved in small volume of extraction buffer and fractionated on sephadex G-10 chromatography which was performed as described by Rajiv Bharadwaj and Chandrashekharaiah (2017). The faction volume was 2.0 ml with a flow rate of 10 ml/hr and 0.025 M sodium phosphate buffer, pH. 7.0 as elution buffer. Among all the fractions obtained, fractions with inhibitor activity were pooled, concentrated and fractionated on Sephadex G-50 gel chromatography which was performed as described by Rajiv Bharadwaj and Chandrashekharaiah (2017) using 0.025 M sodium phosphate buffer, pH. 7.0 as elution buffer. The flow rate was calibrated to 10 ml/hour and 2.0 ml fractions were collected. The inhibitor fractions were further fractionated on RP-HPLC in Reversed-phase octadecylsilica (C18) column in binary solvent system with binary gradient capability and a UV detector. Buffer A is 0.1% (v/v) TFA in water and Buffer B is 100% acetonitrile containing 0.1% (v/v) TFA.

Determination of total protein: TotalProtein was estimated from the inhibitor extract, fraction of sephadex G-10 and G-50 chromatography according to the method of Lowry et al., (1951).

Trypsin and trypsin inhibitor activity

Trypsin activity was measured according to the modified photometric method of Kakade et al., (1969) using the substrate BAPNA. BAPNA (40mg) was dissolved in 2 mL of dimethylsulfoxide (DMSO) and then diluted (1:100) in 50 mM Tris-HCl buffer, pH 8.2 containing 20 mM CaCl₂, prior to enzyme assay. The enzyme assay was carried out by incubating trypsin with BAPNA for 10 min at 37 °C followed by the addition of 30 % acetic acid to arrest the reaction. The absorbance of p-nitroanilide liberated was measured at 410 nm against an appropriate blank. One trypsin (TU) unit is arbitrarily defined as an increase in absorbance of 0.01 at 410 nm under conditions of assay.

The trypsin inhibitor activity was determined by incubating trypsin with an aliquot of inhibitor inhibitor for 10 min at 37 °C. The reaction was started by the addition of BAPNA followed by incubation for 10 min and the reaction was arrested by the addition of 30 % acetic acid. The residual trypsin activity was measured 410 nm against an appropriate blank. The trypsin inhibitory unit (TIU) is defined as the number of trypsin units inhibited under the assay conditions.

Polyacrylamide gel electrophoresis:

An anionic disc gel electrophoresis was carried out essentially according to the method of Davis and Ornstein (1964). A discontinuous gel system consisting of 8% separating gel and4% spacer gel was used. The electrophoresis was carried out in cold applying a current of 20 – 25 mA for 4 hours using tris – glycine (pH 8.3) as electrode buffer and bromophenol blue as marker dye. After the electrophoresis, the proteins were stained with CBB R – 250 for 1 hour and distained using 7 % acetic acid.

Effect of pH and temperature

Determination of pH stability

The effect of pH on the activity of the partially purified Senna alata trypsin inhibitor was studied as described by Chandrashekharaiah (2013) using the buffers, Glycine – HCl buffer (0.2M, pH 2), Sodium acetate buffer (0.2 M, pH 4), Sodium citrate buffer (0.2 M, pH 5.5), Sodium phosphate buffer (0.2 M, pH 6.5), Tris – hydrochloride buffer (0.2 M, pH 8.0), Sodium borate buffer (0.2 M, pH 10). The pH stability was determined by preincubating the partially purified Senna alata trypsin inhibitor with above buffers for 30 min. Trypsin inhibitor assay was performed as described earlier.

Determination of temperature stability

The effect of temperature on the activity partially purified Senna alata trypsin inhibitor was studied at different temperatures ranging between 0 - 90 ºC as described by Chandrashekharaiah (2013). The temperature stability of partially purified Senna alata trypsin inhibitor was studied by pre-incubating, the trypsin inhibitor at different temperatures (0 - 90 ºC) for 30 min. The incubated samples were rapidly cooled and assayed at room temperature. Trypsin inhibitor assay was performed as described earlier.

RESULTS AND DISCUSSION:

The protease inhibitors from the seeds of Senna alata were purified employing salt fractionation, sephadex G-10 and sephadex G-50 chromatography and RP-HPLC. The proteins precipitated from inhibitor extract were loaded onto a Sephadex G-10 column. The proteins were eluted using 0.025 M sodium phosphate buffer, pH 7.0 with a fraction volume of 2 ml. Three peak fractions of proteins were obtained, fraction-I, fraction-II and fraction-III. Among three fractions obtained, fraction – I and II contained protease inhibitor activity. The Sephadex G-10 fraction-II (Fig. 1) containing inhibitor activity was pooled, concentrated and applied on to a Sephadex G-50 column. The proteins were eluted with the buffer and fractions of 2.0 ml were collected at a flow rate of 10ml/h. The protease inhibitor activity was eluted in a two peak fractions, fraction-I and II (Fig.2). The fraction-II containing inhibitor activity was further purified by RP-HPLC (Fig.3.). Two Senna alataprotease inhibitors (SAPI-I and SAPI-II) were purified. SAPI-I was purified to 36.11 fold with a recovery of 42.80% and showed a specific inhibitor activity of 12.16. MUPI-II was purified to 41.23 fold with a recovery of 51.26 and showed a specific inhibitor activity of 19.61. Several trypsin and chymotrypsin inhibitors from various food plants such wheat, soybean, maize, chick pea, potato etc., has been isolated and studied (Richardson, 1977). Two protease inhibitors with a molecular weights in the range of 41,000 and 23,000 Da were isolated and purified from tomato plants (Gregory et al, 1982). Another protease inhibitor from Moringa oleifera having a molecular weight of 23,600 Da was purified on a Sephadex G-75 chromatography ( Bijina et al, 2011). David et al (1977) successfully isolated and purified five low molecular weight protease inhibitors ranging from 6800 to 8000 Da from soybean. Another protease inhibitor was purified from the seeds of Dimorphandra mollis having a molecular weight of ~20 kDa with a single polypeptide chain (Maria et. al., 2000). A 14 amino acid residue with protease inhibitor activity was isolated and studied from seeds of sunflower.

Effect of pH and temperature

The inhibitory activities of purified Senna alata protease inhibitors SAPI-I and SAPI-II from the seeds was found to be stable in the pH range 5 - 8. Similarly, effect of temperature on the inhibitors showed that both were stable between 4 – 65^oC. Scarafoni et al. (2008) purified trypsin inhibitor from saga seeds which showed significant change in the inhibitory activity after subjecting to various pH and temperature conditions.

Fig.1: Separation of Protease inhibitors from the seeds of Senna alata on Sephadex G-10 Chromatography

Fig.2: Separation of Protease inhibitors from the seeds of Senna alata on Sephadex G-50 Chromatography

Fig. 3: Chromatogram showing the purified fractions of protease inhibitors, SAPI-I and SAPI-II on RP-HPLC acquired at 280 nm.

CONCLUSION:

Two protease inhibitors (SAPI-I and SAPI-II) were isolated and purified from the seeds of Senna alata. Both the inhibitors were found to be stable at pH range of 5 - 8 and temperature stable between 4 – 65^oC. These inhibitors inhibited trypsin activity effectively and can be used for therapeutic applications.

ACKNOWLEDGEMENTS:

The authors are thankful to Mangalore University for providing research facilities to carry out this work.

REFERENCES:

1. Amrit Pal Kaur, Satwinder K. Sohal. Pea protease inhibitor inhibits protease activity and development of Bactrocera cucurbitae. Journal of Asia-Pacific Entomology. 2016; 19(4): 1183-1189.

2. Bijina B, Sreeja Chellappan, Soorej M Basheer,. Elyas KK, Ali H Bahkali, Chandrasekaran M. Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization. Process Biochemistry. 2011; 46(12):2291-2300.

3. Caroline BF Mourão, Elisabeth F Schwartz. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals. Mar. Drugs. 2013; 11(6): 2069-2112.

4. Chandrashekharaiah KS. Physico-Chemical and Antifungal Properties of Trypsin Inhibitor from the Seeds of Mucuna Pruriens. Orient J Chem 2013; 29(3): 1061 – 1070.

5. Rajiv Bharadwaj P, Chandrashekharaiah KS. Characterization of Amylase Inhibitor from the Seeds of Mucuna utilis. IOSR Journal of Pharmacy. 2017; 9(1):46 – 51.

6. Davis BJ, Ornstein L. Disc Electrophoresis–2, Method and Application to Human Serum Protiens. Annals of the New York Academy of Sciences. 1964; 121 (2): 404-427.

7. Gregory Plunkett, Donald F Senear, Glen Zuroske, Clarence A Ryan. Proteinase inhibitors I and II from leaves of wounded tomato plants: Purification and properties. Archives of Biochemistry and Biophysics. 1982; 213(2):463-472.

8. Hamao Umezawa. Structures and activities of protease inhibitors of microbial origin. Methods in Enzymology. 1976; 45: 678-695.

9. Jarinyaporn Naowaboot, Supaporn Wannasiri. Anti-lipogenic effect of Senna alata leaf extract in high-fat diet-induced obese mice. Asian Pacific Journal of Tropical Biomedicine. 2016; 6 (3):232-238.

10. Jennifer E Koblinski, Mamoun Ahram, Bonnie F Sloane. Unraveling the role of proteases in cancer. Clinica Chimica Acta. 2000; 291(2): 113-135.

11. Hennebelle T, Bernard Weniger, Henry Joseph, Sevser Sahpaz, François Bailleul, Senna alata. Fitoterapia. 2009; 80(7): 385-393.

12. Kakade ML, Simons NR, Liener IE. The evaluation of natural vs Synthetic substrates for measuring the antitryptic activity of soybean samples. Cereal Chemistry. 1969a; 46: 518-526.

13. Liener IE and Kakade ML. Protease inhibitors. 1980; In: Liener, I.E., Ed., Toxic Constituents in Plant Feedstuff, Academic Press, New York, 7-71.

14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951; 193 (1): 265–275.

15. Maria Lı́gia R. Macedo, Daniela Gaspar G. de Matos, Olga L.T. Machado, Sérgio Marangoni, José C. Novello, Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties, In Phytochemistry, Volume 54, Issue 6, 2000, Pages 553-558, ISSN 0031-9422.

16. Richardson M. The proteinase inhibitors of plants and micro-organisms. Phytochemistry. 1977; 16(2):159-169.

17. Scarafoni, Alessio · Consonni, Alessandro· Galbusera, Valerio · Negri, Armando· Tedeschi, Gabriella · Rasmussen, Patrizia· Magni, Chiara · Duranti, Marcello. Identification and characterization of a Bowman-Birk inhibitor active towards trypsin but not chymotrypsin in Lupinus albus seeds. Phytochemistry. 2008; 69(9): 1820-1825.

18. Tsung-Min Kuo, Gregory Pearce, Clarence A Ryan. Isolation and characterization of proteinase inhibitor I from etiolated tobacco leaves. Archives of Biochemistry and Biophysics. 1984; 230 (2):504-510.

Received on 23.09.2017 Modified on 15.01.2018

Asian J. Research Chem. 2018; 11(2):351-354.

DOI:10.5958/0974-4150.2018.00063.9