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. 2019 Aug 8;29(1):35–43. doi: 10.1007/s10068-019-00638-0

Protein purification from Arachis hypogaea in one step: stability studies and anticarcinogenic analysis

Afaque Ahmad 1, Hirday N Verma 1, Prahalad Bharti 3, Kamlesh Pandey 3, Shahbaz Khan 2, Kapil Dev 2,✉
PMCID: PMC6949355  PMID: 31976125

Abstract

The study involved purification of trypsin inhibitor from the seeds of Indian peanuts (Arachis hypogaea), a member of leguminosae family. The inhibitor was purified to homogeneity via three sequential step procedure i.e., salt precipitation to anion-exchange chromatography. The purity and molecular mass was detected using SDS PAGE analysis i.e. ~ 16 kDa. The purified inhibitor termed as Peanut Trypsin Inhibitor (PTI) which inhibits trypsin belonging to serpins family. Anti- neoplastic potential on breast cancer cells (MCF-7) and normal Human Embryonic Kidney cells (HEK) was determined using MTT assay. PTI exhibited IC50 value of ~ 18.412 µg/mL in HEK cells compared to ~ 9.635 µg/mL in MCF-7 cells. The values were quite comparable to curcumin, the standard anticancer drug demonstrating IC50 values of ~ 21.581 µg/mL and ~ 7.135 µg/mL in HEK and MCF-7 respectively. Therefore, we conclude that PTI may be used as supplement along with the conventional drugs for increased efficacy in the treatment of cancer.

Keywords: Protease inhibitor, Trypsin inhibitor, Arachis hypogaea, Anticancer, Peanut

Introduction

Endogenous proteases are found abundantly in the plants, animals, microbes and aquaculture species which may be the biomarker for many diseases (Wu et al., 2012). Diverse types of protease inhibitors (PIs) have been isolated, characterized, and appraised for their biological potentials. These are small molecular mass peptides capable of inhibiting endogenous proteases. Plant PIs are known to possess various pharmaceutical and biotechnological applications which transfix the attention of researchers. In the course of evolution, plants have evolved supple mechanisms that provide them significant resistance against diverse kinds of unfavorable conditions including insects and phytopathogens (Rakashanda et al., 2013a; 2013b; Ussuf et al., 2001). The PIs may inhibit the growth of pathogenic microbes either by inactivating the specific proteases or by causing the pore formation in the bacterial cell membrane and hence prevent their invasion (Krishnan and Murugan, 2015). In the cell system, the proteases are specifically regulated via various signaling pathways and their dysregulation causes reduction of protease activity and hence lead to several fatal pathologies viz. tumor, arthritis, atherosclerosis, cancer. Various signaling pathways such as cellular interactions, cell growth and activation, cell–cell adhesion, cell cycle, apoptosis, signal transduction etc. are dysregulated due to the non-functioning of these proteases. These dysregulated proteases can be the hallmarks of cancer (Lanza et al., 2004; Rakashanda et al., 2013a; 2013b). PIs occurring naturally which can be obtained from plants, animals and microbes revealed their key role in cancer prevention (Khan et al., 2008). The PIs inhibit their specific proteases by forming strong protease-PI complex which primarily bulge to the proteolysis. Hence, it can be suggested that PIs isolated from plant sources may open the new gateways in the future for cancer treatment (Rakashanda et al., 2013a; 2013b). Several PIs have been isolated from the various plants sources (Mohan et al., 2018; Rakashanda et al., 2013a; 2013b).

Peanut (Arachis hypogaea), a member of leguminosae family, is known to be mostly planted, harvested and consumed crop worldwide. Peanuts seeds were also known to possess oil and protein as the major constituents and hence, it can be served as mostly used food source in various developing countries in order to get rid of diseases caused due to malnutrition (Bobbarala et al., 2009; Sharma et al., 2012). Peanut meal is proved to be nutritionally valuable as the peanuts seeds contain high protein content (47–55%) but the functional properties and activities found in this major part is still uncover and underutilized (Kim et al., 2009; Meulenbroek et al., 2012; Mosolov and Valueva, 2005; Satheesh and Murugan, 2011). Peanuts are also known to be highly rich in nutrients and phytonutrients which are essential and beneficial to human health. The various nutrients found in the peanuts showed that they are an ideal source of protein, fibers, lipids, niacin, fatty acids folate, vitamin E and various essential metal ions viz. magnesium, phosphorus, manganese etc. and also for p- coumaric acid, stilbenoids and stilbenephytoalexins (Cherry, 1977; Sobolev et al., 2006; Yoder et al., 2007). Peanuts are also known to possess the chemical compound resveratrol, which can reduce the risk of cardiovascular diseases and cancer (Chauhan et al., 2012; Sanders et al., 2000). Resveratrol are also a potential source of coenzyme Q10 and of various micronutrients and bioactive compounds responsible for the nutritional and physicochemical properties of peanuts seeds (Bradford and Awad, 2007; Mbagwu et al., 2011; Sivakumar et al., 2006).

Because of these applications of protease inhibitors in medicinal and agricultural fields, peanut become a potential culinary ingredient, thus the search for novel protease inhibitor in peanut has received a keen interest. It was also found that there were very few studies have done on the isolation and purification of PI from Indian peanuts (Arachis hypogaea) (Shigemi et al., 1982). Hence, in the current study, we have purified and characterized the trypsin inhibitor from peanuts (PTI) via single step of purification. Further, we have explored the anticancer potential of PTI in vitro as a part of our continued efforts to explore it for possible development of phytodrug to be used for human benefit. The work has its novelty as this the first report of PTI purification (to the best of our knowledge) to homogeneity and high yield in single step.

Materials and methods

Plant sample and chemicals

Seeds of Arachis hypogaea ‘peanut’ (TPG-41) were obtained from IARI, New Delhi. Trypsin from porcine pancreas, Sodium benzoyl-Dl-arginine-p nitroanilide (BAPNA), and SDS-PAGE chemicals were procured from Sigma-Aldrich and SRL, India and other chemicals were procured from Himedia, CDH, Thermo Fisher and Merck.

Cell lines and cell culture

Human Embryonic Kidney (HEK) and Human Breast Cancer cell line (MCF-7) were maintained in RPMI-1640 grown in 10% fetal bovine serum (FBS), antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). The cells were cultured at 37 °C, 5% CO2 humid condition in CO2 incubator (Thermo Fisher Scientific). All the other chemicals were procured from Sigma Aldrich and Merck.

Screening of the plants sources for the presence of PIs

Edible parts of locally available plants, belonging to families of Malvaceae, poaceae Graminae, Leguminosae, Rutaceae and Solanaceae were taken as the source material for screening protease inhibitory activity.

Selection of potential plant source for the isolation of PI

Those PIs from various plant sources that can be saturated by ammonium sulphate salts were selected for further screening and selection of potential plant source was done. The plant samples which showed more than 60% of protease inhibition was selected and the crude extract was prepared for the isolation of PI as described in the following sections. These sources included A. hypogaea ‘Indian Peanut’.

Purification of peanut trypsin inhibitor

Homogenization and defatting of Arachis hypogaea seeds

Seeds of peanuts were peeled, washed thoroughly in distilled water and then soaked overnight in the homogenization buffer (20 mM Tris- 2 mM CaCl2, pH 8.0). Using blender, the soaked seeds were crushed and then filtered through a multifold cheese cloth. Further, the protease inhibitors viz. 1% of polyvinyl pyrrolidone (PVP) and 250 mM Phenyl-methyl-sulphonyl-fluoride (PMSF) were added to the homogenate to prevent any kind of proteolytic activity. The whole process of purification was performed at 4 °C. The homogenate was centrifuged at 9000 rpm for 40 min at 4 °C to remove the solid debris and the supernatant was collected. Further, the fat content of peanut seeds were removed by adding glacial acetic acid to drop pH to 4 and kept on stirring for 4 h. It was then again centrifuged followed by maintaining pH to 7 by adding few drops of liquor ammonia.

Ammonium sulphate precipitation and dialysis

The supernatant obtained was further saturated with the salt ammonium sulphate up to 30% which precipitates the proteins present in that supernatant. The sample was again centrifuged and supernatant was collected. The pellet was discarded due to the presence of large proteins. The supernatant was again subjected for the 90% ammonium sulphate precipitation. The pellet after 90% saturation was collected by centrifugation at 9000 rpm for 40 min and dissolved in minimal amount of Tris buffer (pH 8.0) followed by an extensive dialysis in the same buffer for 12–24 h.

Anion-exchange chromatography

The dialyzed sample was filtered using membrane filter of 0.2 μm pore size. Then it was loaded on Hi Trap DEAE FF (1 mL, 7 mm × 25 mm) column (GE Healthcare) pre-equilibrated with Tris buffer, pH 8.0 for the purification of protein by weak anion-exchange chromatography. Binding and elution were done at a flow rate of 0.5 mL/min which was controlled by Akta purifier. The column bound protein was washed with equilibration buffer (Tris buffer) which helps in the removal of unbound protein until the absorbance reaches zero at 280 nm. The proteins that remained bound to the column were eluted with NaCl linear gradient (0–1 M NaCl w/v) in the same buffer. The first peak that was eluted at 0.225 M NaCl was pooled, concentrated using Amicon protein concentrator and checked for trypsin inhibitory activity. The fractions showing trypsin inhibition was pooled and run on SDS-PAGE to check the purity of the purified protein. The elution profile was analyzed by unicorn manager (version 5.0) for the absorbance at 280 nm against elution volume (mL).

Trypsin inhibitor activity assay

PTI activity against trypsin was determined by estimating the residual hydrolytic activity of trypsin towards the substrate BAPNA (N-benzoyl-l-arginine-p-nitroanilide) at pH 8.0 after pre-incubation with inhibitor (Erlanger et al., 1961). One trypsin unit is referred as 1 µmol of substrate hydrolyzed per min of reaction. One inhibitor unit was recorded as the quantity of inhibitor needed to inhibit 50% of the corresponding enzyme activity.

Determination of protein concentration

The amount of purified protein and its concentration was estimated by the method of Lowry et al. with some modifications (Lowry et al., 1951). Bovine serum albumin (1 mg/mL) was used as a standard protein to measure the concentration.

Molecular weight determination and purity check by SDS PAGE analysis

Molecular mass of the purified PTI was determined by SDS-PAGE on 12% polyacrylamide slab gel under reducing conditions. PTI was detected by staining the gel with 0.1% Coomassie brilliant blue R-250. The molecular weight standards of 10 to 180 kDa were used for the molecular mass determination. The purity was checked by visualizing the number of bands of sample run.

Stability studies

pH and thermal stability

The PTI was checked for its stability under the extremes of pH condition and determined by incubating it in the buffer of pH range from 2 to 4 h at room temperature. The buffers used was ranging from 1 to 12 at final concentrations of 100 mM: glycine– HCl for pH 1–2, Na-acetate–acetic acid for 3–5, phosphate buffer for pH 4–5, Tris–HCl for pH 7–8, glycine–NaOH for pH 9–12. After incubation at each pH, the residual trypsin inhibitory activities were measured as described earlier.

Thermal stability of PTI was determined in Tris buffer, incubated at various temperatures ranging from 10 to 100 °C (± 0.1 °C) in a water bath for 1 h. The incubated samples were cooled back to room temperature and then centrifuged. The remaining PTI activity against trypsin was measured as described previously.

Effect of detergents, oxidizing and reducing agents

PTI was checked for its stability under the effect of some chemical denaturants (detergents) viz. Triton X-100, SDS and Tween-80 etc. by estimating the % residual trypsin inhibitory activity. The purified PTI was incubated with each detergent for 45 min and the dialysis of that sample was done against Tris buffer. Further, the % residual trypsin inhibitory activity was measured as described earlier.

The PTI was also checked for its stability under the effect of various oxidizing agents viz. Dimethyl sulphoxide and H2O2 and reducing agents (dithiothreitol i.e. DTT and β-mercaptoethanol) on trypsin inhibitory activity of PTI was calculated by incubating PTI with different concentrations of them for 45 min. Further, their % residual trypsin inhibitory activity was estimated as described above.

Determination of in vitro anti-carcinogenic activity of PTI

MTT Assay was carried out as described by Van Meerloo et al. (2011). Cells were seeded at a density of 5 × 103 cells/well in 96-well plate supplemented with 2.5% FBS. After treatment with the PTI, cells were incubated at 37 °C with two different concentrations i.e. 5 μg/mL and 10 μg/mL for 24 h and 48 h. After the required time period, 20 µl of 5 mg/mL MTT solution in PBS (pH 7.4) was added to each well and incubated for 4 h. After that, 150 µl of DMSO was added that dissolves formazan crystals formed by cellular reduction of MTT in wells. The change in absorbance was recorded at 540 nm wavelengths on an ELISA plate reader.

All measurements were done in triplicates. The percent cytotoxicity values were determined by

%Cytotoxicity=AC-AT/AC×100

where (A)T is absorbance of test sample and (A)C is absorbance of control.

Statistical analysis

The experiments were performed in triplicates (n = 3). Graph Pad Prism Software was used to prepare Graphs and to calculate the statistical parameters such as mean, standard error of mean (SEM) etc. The data obtained were expressed as ‘mean ± SEM’.

Results and discussions

Screening of PI from plant sources

Results obtained showed that the plants belonging to other families have more protein content but maximum inhibitory activity was shown only by A. hypogaea belonging to leguminosae family (88.894%). The protein content per 100 g of peanut seeds was also very high (44.986%) as the source belongs to leguminosae family with high nutritive and therapeutic values. Also, due to no colour of the supernatant, the purification was much easier. The protein was found to be much more stable and has high shelf life. As the literature suggested that the PI isolated from garlic, potato, capsicum etc. were thoroughly studied and well characterized earlier (Bhattacharyya et al., 2006) hence we have selected peanut as the novel and potential source for the isolation and purification of PI because there are still no reports and such data available on this plant (to the best of my knowledge).

Purification of PTI

The results of anion exchange chromatography depicted single peak in the chromatogram (Fig. 1A) which was eluted at 0.225 M NaCl showed trypsin inhibitory activity. These fractions were then collected, concentrated using Amicon concentrator which served as the purified protein i.e. peanut trypsin inhibitor (PTI).

Fig. 1.

Fig. 1

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(A) FPLC chromatogram of PTI after ammonium sulphate precipitation followed by dialysis, the dialyzed protein was loaded on Hi Trap DEAE FF column and eluted using gradient of NaCl (0–100% in Tris buffer). The chromatogram represents concentration of PTI eluted (milliabsorbance mA280 at 280 nm) on Y axis with amount of eluted fraction (mL) on Y axis. The second curve represents the gradient of NaCl (0–100% of B) where buffer A is 25 mM Tris–HCl (pH 8.2) and B is 1.0 M NaCl in the same buffer. (B) PTI was run on 12% resolving, 5% stacking SDS PAGE. 20 μl of sample with loading buffer was loaded in each well. The bands were visualized using Coomassie staining. Lane M: Standard molecular marker; Lane 1: purified protein (PTI). (C) Purification profile of PTI calculated at each step of purification

Trypsin inhibitory activity of PTI

The fractions eluted on DEAE column showed trypsin inhibitory activity confirmed that the eluted protein was a trypsin inhibitor. Therefore, we named this eluted protein as Peanut Trypsin Inhibitor (PTI). The specific activity was determined which increased along with the purification process. The purified PTI showed 180180.732 U/mg specific activity with a fold purity of 11.757 and the yield obtained was 12.131% (Fig. 1C). The active fraction thus obtained was used further for various biophysical activities.

Determination of protein concentration of PTI

The amount and concentration of protein was calculated at each step of purification taking BSA as standard. The yield was calculated and the results were summarized in Fig. 1C. The total PTI obtained from 100 g of peanut seeds was 96.81 mg with high homogeneity and strong trypsin inhibitory activity.

SDS PAGE analysis of PTI

PTI was checked for homogeneity and molecular mass by subjected onto the SDS-PAGE and the single band at ~ 16 kDa (lane I) indicated the output with high level of purity (Fig. 1B). The molecular mass was compared to the medium range protein ladder (180 kDa-10 kDa). The purification of PTI was done from the homogenate of peanut seeds (Arachis hypogaea) by three step process which includes salt precipitation, dialysis followed by Anion-exchange chromatography. SDS-PAGE was used to assess the purity and molecular weight of purified PTI. Results obtained in the present study agree to a great extent with those found by other workers (Shigemi et al., 1982). The obtained molecular weight of the PTI i.e. 16 kDa fall in the category of Kunitz type of inhibitors as the literature survey suggest that Kunitz-type inhibitors possess molecular weight in between 16 and 26 kDa (Rodrigues et al., 2003). Hence, the results revealed that the molecular weight of the novel PTI being 16 kDa, it could be placed under the family of Kunitz type inhibitors. Results obtained here also proved that PTI inhibits serine protease such as trypsin so it could also be a member of serine protease inhibitor family.

Stability studies of PTI

pH and thermal stability

In temperature sensitivity studies, trypsin inhibitory activities related with PTI was assayed at temperature ranging from 10 to 100 °C. The optimum temperature lie in range 30–50 °C where activity was recorded to be more than 90%. Residual inhibitory activities were retained remarkably up to 80 °C suggesting its thermal stability nature. Above 80 °C temperature there was a decrease in the inhibitory activities which could be due to denaturation of protein (Fig. 2A).

Fig. 2.

Fig. 2

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(A) Thermal stability studies of PTI was done by incubating it at different temperature range (10–100 °C) for 30 min. PTI was then assayed for % residual inhibitory activity at room temperature. (B) pH stability of PTI at different pH range was observed by incubating it in buffers with pH range 1–12 for 12 h. Further, % residual inhibitory activity was measured

PTI showed maximum residual inhibitory activities at pH 8.0. Further, it maintained optimum activity over mild acidic to alkaline range (pH 6–9) with average activity of ~ 70% with a drop in activity at pH 4. The PTI showed denaturation at extreme acidic and alkaline conditions which may be due to its structural denaturation induced by pH (Fig. 2B).

Further, the stability studies of PTI was performed by incubating it under the extremes of pH, temperature and chemical denaturants viz. various kind of reducing, oxidizing agents and detergents. The literature suggested that intra molecular disulphide bridges found in the PTI might be responsible and required for its functional stability (Kridric et al., 2002). During harsh conditions, these bridges might be broken that causes the functional disability of the PTI. Thermal denaturation of PTI was examined at different temperatures ranging from 10 to 100 °C. As result shown in Fig. 2A, the PTI was found to be fairly thermostable. The results retrieved from pH stability studies of PTI demonstrated that the PTI was quite stable in a pH range of 5.0–10.0. However, there was a drop in PTI activity around pH 4–5 and also at extreme acidic and alkaline conditions. The PTI showed denaturation at extreme acidic and alkaline conditions which may be due to its structural denaturation induced by pH. Residual inhibitory activities were retained remarkably up to 80 °C suggesting its thermal stability nature. Above 80 °C temperature there was a decrease in the inhibitory activities which could be due to denaturation of protein and the inhibitor protein was totally inactive at 100 °C (~ 19% inhibition). This is due to PTI thermal degradation at temperature more than 50 °C. These observations indicate that the PTI has high intrinsic stability in its native state, which gives a high degree of thermal stability. This property of protease inhibitor is typical of all trypsin inhibitor family. Soybean trypsin inhibitor, purified by gel permeation chromatography, had much thermal stability and the presence of a protein substance accelerated the thermal inactivation of the inhibitor (Ellenreider et al., 1980). The results obtained here proven the highly stable nature of PTI which could make it a suitable candidate for use as biopesticides. The midgut of insects and pests possess various kinds of serine proteases responsible for the digestion of food material and a highly alkaline medium. Therefore, the highly stable nature of PTI can easily combat these conditions and can act as naturally occurring pesticide by inhibiting the midgut proteases. Mostly PIs are proven to exhibits anti-feedent properties reported due to their exertive effect against the serine proteases viz. trypsin and chymotrypsin (Ryan, 1990). It might be assumed that the thermal inactivation of proteins may be due to the denaturation or unfolding of them in the presence of intense heat which primarily result in the disturbance of covalent and non-covalent interactions (Lawton and Doonan, 1998). A number of proteinase inhibitors from plants have been reported to have high stability and fair activity up to 70 °C (Mohanraj et al., 2019). Here, the results suggested the thermostablity of PTI under the intense temperature and these eminent stabilities of PTI warrant its applications in various pharmaceutical and nutraceutical industries. High thermal stability of PTI is desirable and required for various biotechnological relevance because it might increases the proteins adequacy and making it the essential source for the commercial exploitation (Pandhare et al., 2002).

Effect of detergents, oxidizing and reducing agents

Ionic and nonionic detergents except SDS have negative impact on TI activities, i.e., 1% SDS increased the residual activities to 135.32 ± 3.464% of PTI when compared to the control. Triton X 100 decreased the inhibitory activities remarkably i.e. 54.485 ± 3.543%. 41.184 ± 5.234% residual activity was observed for 1% Tween-80 (Fig. 3).

Fig. 3.

Fig. 3

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Effect of detergents on trypsin inhibitory activity of PTI by incubating it with detergents for 30 min at room temperature. PTI was then assayed for % residual inhibitory activity at room temperature

Oxidizing agents reduced trypsin inhibitory activities gradually with concentration, i.e., 0.5 mmol/L of DMSO, decreased the activities to 36.912 ± 2.378% while, at 5 mmol/L it was observed up to 3.415 ± 1.061%. Similarly, PTI activity was found to be 15.529 ± 3.966% with 5 mmol/L H2O2 (Fig. 4).

Fig. 4.

Fig. 4

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Effect of oxidizing agents on inhibitory activity of PTI by incubating it with oxidizing agents for 30 min at room temperature. PTI was then assayed for % residual inhibitory activity at room temperature

The residual inhibitory activities of PTI decreased with β-mercaptoethanol. For example, PTI activity with 0.5 mmol/L 2-ME was 96.004 ± 2.491%; and at 5 mmol/L 2-ME, it was 79.593 ± 4.434% respectively. Meanwhile, DTT (0.5 mmol/L) for 3 h on the residual inhibitory activities of PTI was 94.204 ± 4.879%, while with 5 mmol/L DTT; it retained the activities up to 15.085 ± 3.91% (Fig. 5).

Fig. 5.

Fig. 5

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Effect of reducing agents on inhibitory activity of PTI by incubating it with reducing agents for 30 min at room temperature. PTI was then assayed for % residual inhibitory activity at room temperature

Detergents are commonly involved in protein solubilization from lipid membranes and also sustain the solubility of proteins in solution. The increase in PTI activity in presence of SDS as compared to control suggests that it acts as stabilizer for PI. Due to the hydrophobic nature of SDS, rearrangement of peptide backbone conformation might have occurred which would have lead to helix-formation with more hydrophobic residues exposed and hence available to interact with the detergent (Bhattacharyya et al., 2006). On the other hand, decrease in PTI activity with Triton X100 and Tween 80 might be due to multiple factors such as (1) binding of protein with detergent molecules without major conformational change (2) association with specific binding points of native proteins (3) binding with conformational changes in the protein such that most of the hydrophobic residues are exposed for linking with the detergent. Loss of function without a concomitant change in structure proposed that certain essential amino acids responsible for inhibiting enzyme activity and the reactive site residues were affected (Choi et al., 2005).

Moreover, reducing agent such as DTT also affected the residual inhibitory activity of PTI in concentration dependent manner. DTT being strong reducing agent drastically affected the activity of PTI whereas β-ME being weak reducing agent did not affect the residual inhibitory activity of PTI much. Further, the study performed to determine the effect of the detergents on the PTI stability also proven its highly stable nature. Detergents are frequently causes the solubilisation of proteins found in the lipid membranes by enduring the solubility of proteins in the solution. The results revealed in this study concluded that the activity of PTI was quite higher in the presence of SDS as compared to the control which proved that the SDS acts as stabilizer for the purified PTI. The effect of oxidizing agent on the PTI activity was also proved that has been suggested that oxidation can be a key element for PTI activity regulation. Several researches showed that methionine oxidation is the primarily used for regulation of protein activity (Krishnan and Murugan, 2015).

Anti-carcinogenic activity of PTI

This study has reported the anti-neoplastic properties of PTI for the first time in MCF-7 cells (to the best of our knowledge). Our results clearly proved that the PTI was found active against tumor cells when compared to non tumor cells in time and concentration dependent manner. PTI was added in two different concentrations and the cells were incubated for two different time intervals and cytotoxic effect of PTI on cells was assessed. MTT endpoints suggested that PTI showed higher cytotoxicity on MCF-7 cells as compared to normal HEK cell line in time and concentration dependent fashion. PTI exhibited IC50 values of ~ 18.412 µg/mL in HEK cells compared to ~ 9.635 µg/mL in MCF-7 cells. The values were quite comparable to curcumin, the standard anticancer drug demonstrating IC50 values of ~ 21.581 µg/mL and ~ 7.135 µg/mL in HEK and MCF-7 respectively. Hence, we can conclude that PTI shows higher cytotoxicity against MCF-7 cells as compared to HEK cells in time and concentration dependent manner (Fig. 6). Proteases are known to play a regulatory role in various pathologies including viral infections, cancer, thromboembolic disorders, pancreatitis and many others. To combat these pathologies, one can target these proteases by using naturally obtained protease inhibitors. Various PIs obtained from Lavatera cashmeriana have been found to strongly inhibit chymotrypsin, trypsin and elastase. These proteases are responsible for the tumour invasion and metastasis. Our purified PTI inhibits one of these protease i.e. trypsin which indicates its possible anticancer effects (Rakashanda et al., 2013a; 2013b). Rakashnada et al., (2013a; 2013b) reported the IC50 values of Lavatera cashmeriana Protease Inhibitors (LCPI) to be 36 ± 2 μg/mL in Human lung cancer cell line (NCIH322) which was quite higher than the results obtained in our studies (Rakashnada et al., 2013a; 2013b). Hence, PTI demonstrates more inhibitory effect on cancer cell lines and therefore, can be depicted as an anti-tumor drug in near future.

Fig. 6.

Fig. 6

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The effects of the PTI on the proliferation of HEK and MCF-7 cells. Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) FBS, and antibiotics, at 37 °C in a humidified atmosphere containing 5% CO2. Confluent cells (5 × 103cells/well) were treated with PTI at final concentrations of 5 μg/mL and 10 μg/mL for 24 h and 48 h. The % cytotoxicity was determined using the MTT assay

In conclusion, PTI may possess the potential application to act as anticancer agents by showing cytotoxicity in the breast cancer cells. Hence, the detailed mechanism can be investigated that how PTI exerts cancer prevention and act as natural anticancer agent in the cancer treatment thus opening the future gateways for further research.

Acknowledgements

The authors are thankful to Central Instrumentation Facility, Department of Biotechnology, Jamia Millia Islamia, New Delhi to allow the access to instruments for the research work.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Afaque Ahmad, Email: afaquekhan00@gmail.com.

Hirday N. Verma, Email: vc@jnujaipur.ac.in

Prahalad Bharti, Email: bharti.pbh@gmail.com.

Kamlesh Pandey, Email: kamleshpandey.aiims@gmail.com.

Shahbaz Khan, Email: shahbaz.k30@gmail.com.

Kapil Dev, Email: kdev@jmi.ac.in, Email: kapil1814db@gmail.com.

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