A 2% rabbit blood cell suspension was prepared as previously described (13). Using a total of 6 wells of type V blood clots, 25 μL of 0.9% saline was successively added to each well using a pipette gun. Twenty-five microliters of M. oleifera leaves protein was added to the first well of each row. Double dilution was performed (except in the last well of each row, which was used as a blank control), and 25 μL of 2% rabbit red blood cell suspension was then added to each well. The plate was shaken on a micro-oscillator for 1 min to ensure that the mixture was fully mixed. Hemagglutination was observed visually after 2 h at room temperature and denoted by 2ⁿ (where n is the number of holes).

Lyophilized M. oleifera leaves protein was dissolved in PBS (pH 7.4, 0.01 mol/L) to obtain a solution with an initial concentration of 5 mg/mL. The solution was heated in a water bath, and the temperature was increased starting from 20°C and maintained constant for 5 min every 10°C. The M. oleifera leaves protein solution was removed, cooled at room temperature, and diluted in a 96-well “V” type hemagglutination plate. The hemagglutination activity was measured 2 h later.

The test sugar was prepared in 0.5 mol/L mother liquor, 50 μL was added to well No. 1 of a 96-well “V” type hemagglutinin plate, and 50 μL of PBS buffer (pH 7.4, 0.01 mol/L) was added to wells No. 2–9. To each well,50 μL of Moringa leaves protein solution was added, and after 30 min at room temperature, 50 μL of red blood cell suspension was appended. The plate was allowed to stand for 2 h before the hemagglutination activity was measured. The following saccharides were used: α-methyl-D-glucopyranoside, maltose-hydrate, D-mannose, D(+) anhydrous glucose, D-raffinose pentahydrate, methyl-α-D-glucopyranoside, lactose, and methyl-β-D-galactoside.

Moringa oleifera leaves protein solution was prepared with 0.01 mol/L PBS buffer (pH 7.4) and set aside. Fifty microliters of 11ion solutions (0.1 mol/L) were added to each well of the 96-well “V” hemagglutination plate and then diluted with PBS buffer (pH 7.4, 0.01 mol/L). Subsequently, 50 μL of Moringa oleifera leaves protein solution was added to each well, and the hemagglutination activity was detected.

Moringa oleifera leaves protein solution with an initial concentration of 5 mg/mL was prepared in a series of buffer systems with pH values from 3.7 to 13.0. The solution was left at room temperature for 2 h, and the hemagglutination activity under different pH conditions was measured after multiple dilution. The following buffers were used: NaCl-HAc buffer (0.1 mol/L) at pH 3.7–5.8, 0.1 mol/L NaH₂PO₄-Na₂HPO₄ buffer at pH 6.2–7.8, 0.1 mol/L Tris-HCl buffer at pH 8.3–9.1, 0.1 mol/L Na₂CO₃-NaHCO₃ buffer at pH 9.5–10.8, 0.1 mol/L Na₂HPO₄-NaOH buffer at pH 11.2–11.9, and 0.1 mol/L KCl-NaOH buffer at pH 12.2–13.0.

The hemolytic activity was determined using the assay (19). Briefly, PBS-washed rabbit erythrocytes were prepared in 0.1 M PBS (pH 7.2, 1:9 v/v) containing 10 mM calcium chloride. Fifty microliters of various concentrations of M. oleifera leaves protein (5, 10, 20, and 40 mg/mL) was incubated with 950 μL of rabbit red blood cells (1:20 v/v) and centrifuged (2,000 rpm, 5 min). The absorbance of the supernatant (erythrocyte lysate) was read at 540 nm using a microplate reader. Erythrocytes incubated in PBS (pH 7.2) were used as a negative control, and 20% Triton X-100 was used as a positive control. The percentage of hemolysis was calculated using the following formula:

where a is the percentage of hemolysis, Z is the erythrocyte hemolysis rate, At is the absorbance of the test group, Anc is the absorbance of the negative control group, and Apc is the absorbance of the positive control group.

M. oleifera leaves protein (20 mg/mL) was incubated with 0.2 M maltose-hydrate, α-methyl-D-glucopyranoside, D-mannose, D(+) anhydrous glucose, D-lactate pentahydrate, lactose and methyl-β-D-galactoside for 2 h at 25°C and then with 10% freshly prepared rabbit red blood cell suspension (200 μL) at 30-min intervals with gentle shaking for 4 h. The samples were centrifuged (2,000 rpm, 5 min), and the absorbance values of red cell lysates were analyzed at 540 nm. To calculate the percentage of hemolysis, erythrocytes incubated in PBS (pH 7.2) were used as a negative control, and 20% Triton X-100 was used as a positive control.

M. oleifera leaves protein (20 mg/mL) was incubated with MgCl₂, KCl, FeCl₃, CaCl₂, NaCl, FeCl₂, NH₄Cl, BaCl₂, and AlCl₃ stock solutions for 2 h at 25°C and then with 10% freshly prepared rabbit red blood cell suspension (200 μL) with gentle shaking at 30-min intervals for 4 h. Samples were centrifuged (2,000 rpm, 5 min), and the absorbance values of red cell lysates were analyzed at 540 nm. To calculate the percentage of hemolysis, erythrocytes incubated with PBS (pH 7.2) were used as a negative control, and 20% Triton X-100 was used as a positive control.

With an extraction time of 6 h, a saturation level of ammonium sulfate equal to 60%, and a solid-to-liquid ratio of 1:8, the effect of the type of extract on the protein extraction rate of M. oleifera leaves is shown in Figure 1A. The protein extraction rates of the Tris-HCl and PBS groups were significantly higher than those of the other groups, and that of the PBS group was higher. This finding may be related to the strong buffering effect of PBS buffer, which can better maintain the structure and activity of the protein; this finding is consistent with the results reported (14). Therefore, PBS was selected as the optimal extract for M. oleifera leaves protein.

The effect of extraction time on the extraction rate of M. oleifera leaves protein was studied under the condition of 60% ammonium sulfate saturation and solid-to-liquid ratio of 1:8. As shown in the Figure 1B, an increase in the extraction time from 2 to 6 h increased the extraction rate of M. oleifera leaves protein from 5.57 to 11.3 mg/g. However, increasing the extraction time to 10 h decreased the protein extraction rate. This finding was probably observed because intracellular protein-degrading enzymes also are dissolved during the longer extraction time, and these protein-degrading enzymes degraded part of the dissolved proteins, thereby reducing the protein extraction rate (15). The optimal M. oleifera leaves protein extraction rate of 11.3 mg/g that was obtained with an extraction time of 6 h, which was 2.03 times that obtained with an extraction time of 2 h.

Next, the effect of solid-liquid ratio on the protein extraction yield of M. oleifera leaves was explored at an extraction time of 6 h and ammonium sulfate saturation of 60%.leaves. As shown in the Figure 1C, an increase in the liquid-to-solid ratio from 1:4 to 1:8 increased the protein extraction rate of M. oleifera leaves from 10.89 to 15.96 mg/g. This rapid enhance in the protein extraction rate may be due to an appropriate growth in the solvent volume to improve the contact area for M. oleifera and augment its dispersion, which enhances the diffusion effect and thus advance the extraction rate (16). However, with a further increase in the solid-to-liquid ratio to 1:12, the protein in plant cells becomes completely dissolved, and the amount of solvent has no effect on the protein extraction rate (15). The results showed that once the soluble protein concentration reached its maximum value, an increase in the solid-to-liquid ratio decreased the protein concentration. Therefore, the optimal solid-to-liquid ratio for extracting M. oleifera leaves protein was determined to equal 1:8.

The effect of ammonium sulfate saturation on the extraction rate of M. oleifera leaves at a solid-to-liquid ratio of 1:8 with an extraction time of 6 h is shown in Figure 1D. An increase in the ammonium sulfate saturation level from 40 to 60% raised the extraction yield of M. oleifera leaves protein from 10.03 to 14.37 mg/g, which indicated that M. oleifera leaves protein mainly consisted of salt-soluble proteins. However, further improve in the saturation level of ammonium sulfate increased to 80% decreased the extraction rate of M. oleifera leaves protein by 0.28 times compared with that obtained with an ammonium sulfate saturation level of 60%; this finding may be due to the salt-out of protein in a solution containing a high salt concentration, which would reduce the extraction rate of M. oleifera leaves protein (17). Therefore, the optimal ammonium sulfate saturation level for M. oleifera leaves protein extraction was determined to equal 60%.

As shown in Table 4, when the temperature was below 60°C, the M. oleifera leaves protein was very stable, maintaining its full hemagglutination activity, and the hemagglutination titer was 2⁸. Increasing the temperature gradually decreased the agglutination activity of M. oleifera leaves protein until a temperature of 110°C, which resulted in almost complete loss of hemagglutination activity. These findings show that temperature has a marked effect on M. oleifera leaves protein. The hemagglutination activity could be maintained under cooling conditions but might be reduced or even completely lost by increasing the temperature (28, 29).

The inhibitory effects of eight sugars on M. oleifera leaves protein are shown in Table 5. The hemagglutination titer of α-methyl-D-glucopyranoside was 2², and those of maltose-hydrate, D-mannose, D(+) anhydrous glucose and were 2², 2³, 2⁰, and 2⁴, respectively. In addition, the hemagglutination titer of methyl-α-D-glucopyranoside was 2³, that of lactose was 2², and that of methyl-β-D-galactoside was 2⁴. In the reactive state of hapten inhibition, the sugar with the best inhibitory effect is called the specific sugar of lectins. The results showed that D(+) anhydrous glucose was the specific inhibitory sugar of M. oleifera leaves lectin because it can better inhibit the agglutination reaction of rabbit red blood cells and M. oleifera leaves protein. α-Methyl-D-glucopyranoside, lactose and maltose-hydrate also have a partial inhibitory effect on the hemagglutination reaction (30–33).

Ions exert an important effect on protease activity and thermal stability (34, 35, 39). Most legume lectins are ions -binding proteins or glycoproteins. Ions stabilize the structure and increase the resistance of lectins to heat and thus inactivate proteolytic enzymes (40). The effect of ions on the hemagglutination activity of M. oleifera leaves protein was determined, and the results are shown in Table 6. As revealed by the results, M. oleifera lectin is aion-dependent lectin. K⁺, Na⁺, Mg²⁺, and NH⁴⁺ induced loss of the hemagglutination activity of M. oleifera, whereas M. oleifera leaves protein was found to be dependent on Ca²⁺, Fe³⁺, Fe²⁺, and Al³⁺. After Ca²⁺, Fe³⁺, Fe²⁺, and Al³⁺ treatment, the protein continued to exhibit high hemagglutination activity, and its hemagglutination titer was 2⁵-2⁷.

Lectins are generally more stable to acidic and basic conditions (41). Different pH values have obvious effects on the hemagglutination activity of M. oleifera leaves protein. The experimental results are shown in Table 7. The highest hemagglutination activity of M. oleifera leaves protein was observed at pH 3.7, and the hemagglutination activity of M. oleifera leaves protein was completely lost at a pH value of 11.9. The reasons for this finding are as follows: at near-neutral pH, the calm electric repulsion energy is smaller than the energy of other stable protein interactions, and the M. oleifera leaves protein is thus stable (42, 43). However, the strong molecular electrostatic repulsion caused by the high electrostatic charge at extreme pH leads to the swelling and expansion of M. oleifera leaves protein molecules and the ionization of carboxyl, phenolic hydroxyl and sulfhydryl groups partially buried in the protein molecules (43, 44). These ionic groups expose themselves to the water environment, resulting in the dispersion of polypeptide chains, which fundamentally changes the spatial structure of M. oleifera leaves protein and thus leads to irreversible denaturation and loss of vitality.

The erythrocyte is considered a simple mimic of the endosome membrane and is thus a model for the study of protein-membrane interactions and for the in vitro assessment of the biocompatibility of important biotherapies (36). Intravenous therapeutic agents can trigger hemolysis, which is characterized by the release of hemoglobin into plasma as a result of the destruction, interference, or breakdown of red cells, which makes the search for specific proteins as carrier molecules imperative (37, 38). As shown in Figure 4, the cytocompatibility and endolytic activity of M. oleifera leaves proteins on rabbit red blood cells were studied using the hemolysis test, and the percentage of hemolysis was found to increase in a concentration-dependent manner. The hemolysis rate of M. oleifera protein at a concentration of 20 mg/mL was 5.68%, whereas that of the control group was 0.1% (P < 0.001).

As shown in Figure 5, a significant difference in the hemolysis rate was found between M. leaves-treated erythrocytes and erythrocytes preincubated with α-methyl-mannoside, galactoside, and raffinose (P < 0.001). The strong inhibitory activity of lactose may indicate that the lectin possesses an extended binding site. Furthermore, the decreased hemolytic activity of M. oleifera leaves protein in the presence of α-methyl-mannoside, galactoside, and raffinose further confirmed the previously demonstrated fact that M. oleifera leaves lectins have an affinity for ketose sugars, which inhibits the hemolytic activity of M. oleifera leaves protein. To improve the efficiency and reduce the side effects of specific therapeutic agents, protein-related targeted drug delivery systems have become a favorable strategy (18, 19).

As shown in Figure 6, among K⁺, Na⁺, Mg²⁺, NH⁴⁺, Fe²⁺, Ca²⁺, Fe³⁺, and Al³⁺ ions, the higher hemolysis rate was observed with erythrocytes treated with Fe²⁺ and M. oleifera leaves protein, indicating that Fe²⁺ caused erythrocytes to be destroyed, interfered or decomposed and released into plasma. However, Al³⁺ inhibited the hemolytic activity of M. oleifera protein, and similar results were reported for calcium-dependent hemolysin purified from Cucumis acanthopanthus (20) and Granella anadarensis (21).