Purification, Partial Characterization, and Evaluation of the Antiulcer Activity of Calotropis procera Leaf Lectin

Abstract

Background: Lectins are proteins with therapeutic and diagnostic potential that can be applied in battling various ailments.

Aim and objective: This study was designed to purify and characterize the hemagglutinating activity derived from the leaves of Calotropis procera and its possible role in protecting the stomach against ethanol-induced lesions.

Methods: The Calotropis procera leaf lectin (ProLec), was isolated by homogenization of the defatted leaf powder in Phosphate-Buffered Saline (PBS) and purified by affinity chromatography on Sephadex G-100. The lectin was eluted from the affinity column by 3% acetic acid and was physicochemically characterized. In a dose-dependent manner, ProLec was administered to rats with ethanol-induced ulcers, and biochemical, histopathological, and toxicological examinations were performed.

Results: ProLec is a heterodimer of 75 and 68 kDa. It agglutinated all human RBCs, whereas it showed weak interaction with animal erythrocytes. The protein was optimally active at 25 °C and was labile above this temperature. ProLec exhibited two pH optima and was a metalloprotein requiring Ca, Mn, and Ni. It contains 1.6% tryptophan residues of which about 1% is exposed and critical for lectin activity. The lectin exhibited a potent gastroprotective effect against ethanolinduced gastric lesions with no apparent toxicity to both kidneys and liver. Examination of the pH of the gastric juice of lectin-treated animals indicated a possible role of lectin in maintaining stomach acidity within the normal ranges compared to the gastric juice pH of animals that received ethanol only.

Conclusion: These results may suggest that ProLec could conceivably be a good future drug for the treatment of gastric ulcers, however, extensive immunological and toxicological research remains to be done.

Keywords: Calotropis procera; gastroprotective; lectin; medicinal plant; purification; ulcer.

Figure 1A

Extraction of ProLec by using different buffers. Different buffers with variable pHs were used for ProLec extraction, these buffers were: 50 mM PBS pH 7.5 (PBS); 0.145 M, NaCl (N.S); 50 mM Tris-HCl, pH 7.5 (Tris); 50 mM Na-acetate, pH 5 (Acetate); and 50 mM Borate pH 9 (Borate).

Figure 1B

Extraction of ProLec by using PBS pH 7.5 at different molarities 20, 30, 40 and 50 mM

Figure 2A

Elution of ProLec from the affinity resin Sephadex G-100. Six hundred mg of C. procera leaves crude extract was loaded onto the Sephadex G-100 column. The flow throw was recycled at least 3 to 4 times. The column was washed with an ample amount of 40 mM PBS pH 7.5 to remove unbound protein till the OD 280 of the washing dropped to ≤0.02. The bound lectin was eluted with 0.3% acetic acid prepared in 0.150 M NaCl. Fractions were adjusted to neutrality by 0.2 N NaOH and assayed for hemagglutinating activity.

Figure 2B

Native-PAGE crude protein extracted by varying buffers and pure ProLec obtained by affinity on Sephadex G-100. The arrow points to the pure protein. 30 µg protein were loaded and the gel was stained by coomassie brilliant blue G-250.

Figure 2C

SDS-PAGE of pure ProLec obtained by affinity on Sephadex G-100. Lane on the right-hand side indicates the standard molecular weight markers 30 µg protein were loaded and the gel was stained by coomassie brilliant blue G-250.

Figure 3A

Effect of pH on ProLec hemagglutinating activity. Aliquots of lectin were incubated with buffers of different pH values ranging from 2 to 11 at room temperature for 2 hours. 0.1 N HCl or 0.1 N NaOH were used to adjust the pH of the lectin solution to neutrality. The hemagglutinating activity was assayed as shown in the experimental body.

Figure 3B

Effect of different temperature ranges on Prolec agglutinating activity. Thirty µL aliquots of lectin (1 mg/mL) were incubated in a water bath at varying temperatures ranging from 20 to 90 °C with an increment of 10 degrees for 30 minutes. The lectin samples were immediately cooled on ice and the hemagglutination activity was reported.

Figure 3C

ProLec thermal stability at the optimal temperature. ProLec was incubated at its temperature optima (20 °C) for 3hrs. An aliquot was removed every 30 minutes and the hemagglutinating activity was tested as shown in the experimental body.

Figure 4

Effect of metal ion on ProLec hemagglutinating activity. 2 mg/mL lectin solution was dialyzed exhaustively against 50 mM EDTA, followed by dialyzing against double‐deionized water to remove the excess EDTA. EDTA treated lectin was incubated with 2 mM metal ions, Ferrous, Zinc, Mercury, Magnesium, Manganese, and Calcium for 2 hrs at room temperature followed by a hemagglutination assay. EDTA untreated lectin served as a control of 100% activity.

Figure 5

Oxidation of ProLec exposed tryptophan by N-bromosuccinimide (NBS). The ProLec solution at 0.35 mg/mL was prepared in acetate buffer, pH 5.0 was titrated with the freshly prepared NBS (10 mM). Aliquots of NBS was added gradually to the ProLec solution in instalments of 20 µL (0.035 mg) each with shaking. The drop in absorbance at 280 nm as well as the hemagglutination activity were monitored at each point. The percentage of tryptophan residues was calculated essentially as described (Spande and Witkop 1967).

Figure 6

Effect of different doses of ProLec on rat’s stomach: (A): rat received saline; (B): rat received ethanol; (C): rat received pantoprazole; (D): rat received a low dose of ProLec; (E): rat received a medium dose of ProLec; (F): rat received a high dose of ProLec.

Figure 7A

Histopathological analysis of stomach sections of different animals treated groups and controls.

Figure 7B

Histopathological analysis of kidneys and liver sections of rats received 1 mg/Kg ProLec.

Figure 8

The effect of ProLec on the gastric juice pH. The values are given as mean S.E.M. (n = 6). Pantoprazole was used as a positive control. All groups treated with different doses of ProLec followed by absolute ethanol are statistically significant as compared to the group that received only absolute ethanol (p < 0.05).