2.1 Extraction of proteins

The seeds of D. stramonium were collected from local areas of Greater Noida, Uttar Pradesh. Following a distilled water wash, the seeds (Fig. 1) were allowed to air dry and then kept at room temperature. Seeds were grinded and homogenized in an extraction buffer composed of 0.2 M sodium chloride and 50 mM sodium phosphate (PBS; pH 7.4). The homogenate was filtered by using a muslin cloth. The residue was discarded, and the filtrate was used for centrifugation at 10000 rpm for 30 min at 4 °C. After this, the pellet made up of cellular debris was discarded, and the supernatant was used for further protein precipitation (Sachin et al., 2021).

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Fig. 1

Seeds of Datura stramonium (sciencephotogallery.com/featured/thorn-apple-datura-stramonium-seeds).

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2.2 Protein fractionation by ammonium sulphate precipitation method

The supernatant was used to do ammonium sulphate precipitation of proteins in different fractions. Ammonium sulphate was added to the crude sample to make it a 20 % saturated solution and kept overnight for the precipitation of proteins (Wingfield, 2001; Kirar, et al., 2023). Centrifugation was done further at 10000 rpm for 15 mins. at 4 °C. The pellet was dissolved in a 20 mM phosphate buffer, and the supernatant was further used for the addition of ammonium sulphate to make it 60 % saturated and was kept overnight. Centrifugation was done further at 10000 rpm for 15 mins. at 4 °C. The pellet was suspended in a 20 mM phosphate buffer (pH 7.4). The supernatant was further used to add ammonium sulphate to make it 60 % saturated and was kept overnight. Centrifugation was done further at 10000 rpm for 15 mins. at 4 °C. The pellet was suspended in a 20 mM phosphate buffer (pH 7.4). The supernatant was further used to add ammonium sulphate to make it 90 % saturated and was kept overnight. Centrifugation was done further at 10000 rpm for 15 mins. at 4 °C. After that pellet was dissolved in a 20 mM phosphate buffer (pH 7.4), and the supernatant was discarded. Dialysis was done for 20 %, 60 %, and 90 % dissolved pellet fractions to remove (NH₄)₂SO₄ using 20 mM Phosphate buffer (pH 7.4). SDS-PAGE further analyzed the dialyzed sample.

2.3 Purification of targeted protein by ion exchange chromatography

Dialyzed fractions of 20 %, 60 %, and 90 % precipitated proteins were used for purification on anion exchanger-based resin DEAE-Sepharose Fast Flow column chromatography. 4 mL of DEAE resin was packed on a chromatographic column of dimension 40 × 1.5 cm and washed with water. Further, the resin was equilibrated with phosphate buffer pH 7.4. 40 mg/ml dialyzed protein was dissolved in 1 mL of equilibration buffer and loaded onto the column. To remove unbound proteins, protein-bound gel was washed with the equilibration buffer with a flow rate of 300 µl/min. The elution of bound proteins was done using increasing NaCl concentration (100, 200, 300 mM, and 1 M) in 50 mM phosphate buffer with pH 7.4. Eluted fractions were collected and evaluated for hemagglutination activity and for SDS-PAGE. Estimation of the purified sample was done by using Lowry’s methods (Lowry et al., 1951) along with a standard graph prepared by using 1 mg/ml of BSA.

2.4 Hemagglutination assay

Human erythrocytes of B and O blood groups were used to perform hemagglutination assay (13). 100 mM, 200 mM, 300 mM, and 1 M NaCl eluted protein fractions were used to perform hemagglutination assay. Protein fractions were serially diluted in PBS buffer (pH 7.4) on a U-shaped 96 well plate and 2 % erythrocyte suspension was added in all wells. After this incubation was done at 37 °C for 30 min. Hemagglutination titer was calculated as the reciprocal of the maximum dilution of protein which shows complete erythrocyte agglutination (Yagi et al., 2002).

2.5 Periodic acid Schiff’s staining

PAS (Periodic Acid Schiff) stain is the most flexible and commonly used carbohydrate visualization technique. Periodic acid – Schiff (PAS) is a staining method to identify polysaccharides in tissues. These can be glycogen and mucous substances such as glycoproteins, glycolipids, and mucins. 12 % SDS-PAGE was performed with the eluted Datura protein and the standard chick ovalbumin. The gel was immersed in 12 % TCA (tri-chloro acetic acid) for 30 min. as a fixative. TCA was removed and the gel was treated with mili Q water then transferred into the 1 % periodic acid reagent prepared with 3 % acetic acid and incubated for 1 h. After the incubation period, the gel was treated thoroughly with mili Q water for 90 min. with a routine change of water in each 10 min. The gel was transferred into the Schiff’s reagent for 1 h in the absence of light. After 1 h, the gel was washed with distilled water and incubated in 7 % acetic acid for 1 h and the gel was dried for the development of the bands.

2.6 Antiproliferative assay using MTT

Purified protein was tested in vitro against Caco-2 (colon) cancer cell lines to see if it has antiproliferative properties (Monks et al., 1991; Boleti et al., 2008). Optimum concentrations of cells were seeded in 96 well plates until the cell monolayer became confluent. The human cancer lines growing in DMEM with 10 % heat-inactivated FBS at 37 °C, 5 % CO₂ in a CO₂ incubator were treated for 72 hrs. with different concentrations of purified protein. Before treatment, protein solution at final concentrations of 0.25, 0.50, 1.0, 5.0, 10.0, 20.0, and 50.0 µg/ml was prepared in DMEM with heat-inactivated 10 % FBS.

As a control, some wells received the same concentration of cells and no protein in the DMEM medium. The MTT experiment was conducted using DMEM and 5 % fetal bovine serum, per the methodology provided by Heinrich et al., 2005. MTT assay calculates the reduction of yellow MTT by the action of enzyme succinate dehydrogenase to an insoluble, formazan which produces a dark purple colour, inside the mitochondria. In each experiment, 10 µl of MTT was added to the cells for 3 h incubation at 37 °C, 5 % CO₂. Crystals of purple formazan were dissolved in isopropanol without water. At 37 °C, the dissolved purple solution was incubated for three hours. Using a spectrophotometer, the solubilized formazan reagent and 5 % CO2 are measured at 570 nm, with a background absorbance of 690 nm. An average of a few empty wells that contained only MTT solution, but no cells were used to serve as a background control (blank). Since the reduction of MTT occurs in live cells, it is used to measure cell viability.

3.1 Extraction and ammonium sulphate precipitation

The Datura stramonium seed protein was extracted using a phosphate buffer at a pH of 7.4. The homogenized sample was filtered with a muslin cloth, followed by centrifugation at 10,000 rpm for 15 min at 4 °C. Ammonium sulphate ((NH₄)₂SO₄) precipitation was performed using the supernatant. Samples with 20 %, 60 %, and 90 % ammonium sulphate saturation were obtained. The pellet from the 20 %, 60 %, and 90 % saturated sample was re-dissolved in 20 mM phosphate buffer (pH 7.4). Dialysis was performed to remove ammonium sulphate, and then SDS-PAGE was performed to visualize protein bands. Protein bands were observed in the crude sample of 60 % saturated fraction's pellet in the SDS-PAGE gel after staining the gel, as shown in Fig. 2. Therefore, protein purification was carried out from this 60 % fraction using ion-exchange chromatography.

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Fig. 2

A) Elution peak with 200 mM NaCl and b) SDS-PAGE analysis showing crude, unbound, purified protein peak and protein ladder.

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3.2 Protein purification

The dialyzed sample was utilized for anion exchange chromatography. 4 ml of DEAE resin was packed on a chromatographic column of dimension 40 × 1.5 cm and washed with water. The resin was equilibrated by using phosphate buffer pH 7.4. After equilibration 40 mg/ml of sample dissolved in the buffer was loaded onto the column. Unbound was collected from the column with the help of a phosphate buffer.

Bound proteins were eluted using NaCl, increasing the concentration (100, 200, 300 mM, and 1 M) in 50 mM phosphate buffer pH 7.4. A single peak was observed in 200 mM NaCl elute fraction as shown in Fig. 2a. SDS-PAGE revealed the presence of a single band of approximately 30 kDa, as shown in Fig. 2b. The elute peak was further tested for hemagglutination activity. The purified sample was estimated using Lowry’s methods (Lowry et al., 1951) along with a standard graph prepared by using 1 mg/ml of BSA. The concentration of purified protein was approximately 1 mg/ml.

3.3 Hemagglutination assay

Purified protein peak showed hemagglutination activity with human (B and O) erythrocytes as shown in Fig. 3. Hemagglutination titre was calculated as the reciprocal of the maximum dilution of protein which shows complete erythrocyte agglutination. Titre value of hemagglutination was observed at a minimum concentration of 15 µg/ml (B + ) and 3 µg/ml (O + ) of elute peak sample. This analysis predicts the lectin-like properties of purified protein samples. This protein sample showed a molecular mass of approximately 30 kDa on SDS-PAGE analysis. Further, the glycosylation nature of protein was determined by PAS staining.

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Fig. 3

Hemagglutination assay of purified protein using human O and B blood cells.

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3.4 Periodic acid Schiff’s staining

12 % SDS-PAGE were performed with the 200 mM NaCl eluted Datura protein and the standard as a chick ovalbumin (Leite et al., 2012). PAS stain of partially purified datura protein with ovalbumin as a positive standard shown in Fig. 4, the visible bands show the glycosylated nature of the protein which is 30 kDa DSL and the standard Ovalbumin with 42 kDa.

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Fig. 4

Periodic acid Schiff’s staining of purified protein with standard ovalbumin.

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3.5 Cell viability assay by using MTT

To evaluate the anti-cancer property of isolated Lectin, Human cancer Caco-2 cells were treated in vitro conditions with purified protein and cell viability analysis was studied through MTT assay, and the results are shown in Fig. 5. Approximately 53 % growth inhibition of cells were recorded in this study.

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Fig. 5

Cell viability assay of Caco-2 Cell lines treated with varying concentrations of purified protein.

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