2.3.1 LC-MS/ESI-MS analysis

The protein bands were cut separately from the gels, dissolved, decolourized and subjected to trypsin digestion (Shu et al., 2009). The trypsin digested peptides were identified using LC-MS/ESI-MS (Orbitrap XL, Thermo Scientific, Breman). MASCOT search engine (Matrix Science) was used for peptide match and identification.

2.3.2 Sequence alignment

The peptides obtained during LC MS/ESI MS fragmentation were matched with already known RIPs from the protein database as described by Raj and Vennila (2013). Pairwise alignment was carried out with the Protein Information Resource (PIR) alignment tool. The identity and similarity between the peptides and known RIP was tabulated for comparison.

2.4.1 Antimicrobial activity

The bacterial and fungal strains were procured from Microbial Type Culture Collection (MTCC), India and grown in suitable medium for maintainence.

Antimicrobial activity was checked with nosocomial infectious organisms – Gram negative bacteria (Escherichia coli (MTCC 443), Klebsiella pneumonia (MTCC 530) and Pseudomonas aeruginosa (MTCC 799)), Gram positive bacteria (Staphylococcus aureus (MTCC 121) and Bacillus subtilis (MTCC 441)) and fungi (Candida albicans (MTCC 227) and Fusarium oxysporum (MTCC 284)). Agar well diffusion method was adopted (Thomas and Veda, 2007) for evaluation of antimicrobial activity of ARIP. The plates with the media were seeded with overnight grown culture containing 10⁶–10⁷ cells. The purified fractions of ARIP at a concentration of 10, 20 and 30 µg were laid on the wells along with the control. The standard drugs Gentamycin (2 µg) and Cyclohexamide (2 µg) were used as controls for bacteria for fungi respectively. The zone of inhibition (mm) was measured after an overnight incubation.

2.4.2 Antimutagenic activity

The antimutagenic activity was performed by an in vitro Ames assay using Salmonella typhimurium mutant strains (MTCC 1252) as per the protocol specified by Ames et al.(1975). The contents (0.5 ml of ARIP in varying concentration of 0.5 to 2 mg/plate, 0.1 ml of bacterial culture, 0.2 ml of 0.5 mM histidine biotin solution and 0.1 ml of standard mutagen Sodium azide of concentration 1.5 μg/ml) were transferred to 2 ml of molten top agar in a test tube, and mixed thoroughly. This semisolid agar was immediately spread on the layer of minimal glucose agar plates in the petri dish. The dishes were incubated at 37 °C for 48 h and the number of revertants were counted (Issazadeh and Morteza, 2012; Sharififar et al., 2016). A plate containing agar, bacterial strain and the mutagen was considered as positive control. The solvent Dimethyl sulfoxide (DMSO) with agar and bacterial strain was used as a negative control. To detect the indirect mutagenic effect caused by metabolites of the test, liver homogenate of the rat along with the co-factors (Co-factor I- MgCl₂ and KCl and Co-factor II Na₂HP0₄) (known as S9 mix) was used. This experiment was done in triplicates in presence and absence of S9 mix. The antimutagenic potential is calculated based on the percent of inhibition as follows:$$\text{Inhibition}\%=\left(\left[\text{A}-\text{B}\right]\right)/\text{A}\times 100$$where A and B are the number of revertants in each plate, in the presence of positive control and mutagen or/and sample.

2.4.3 Cytotoxic activity

The in vitro cytotoxic activity was assessed using MTT assay (Mosmann, 1983). AGS (Gastric cancer cell lines) was procured from National Centre for cell Science, Pune, India and seeded into 96 well microtitre plates (1.0 × 10⁴ cells /well) along with 100 µl of RPMI-1640 medium followed by 24 h incubation at 37 °C with 5% CO₂. Media was replaced with various concentrations of ARIP and incubated the cells for 48 h. After the measurement of viable cell count, 10 µl MTT solution (5 mg/ml in PBS) was added to the well followed by incubation for 4 h. The formazan crystals were dissolved in 100 µl of DMSO, incubated for 5 min in dark and OD was measured at 570 nm in a microplate ELISA reader. A dose–response curve of sample concentration (μg/ml) versus cell viability (%) was plotted. The IC₅₀ values (concentration that is lethal to 50% of the cells) were calculated (Uthaya Kumar et al., 2018). Ricin and 5 Fluorouracil (Chemotherapeutic drug for Gastric cancer) were used as standards for comparison.

2.4.4 Statistical analysis

SPSS software (IBM Corporation) was used for statistical analysis of the data. The differences with P < 0.05 was considered significant.

3.2.1 LC-MS/ESI-MS

The LC-MS/ESI-MS mass spectra between 9 and 17 min yielded 19 peaks for ARIP-A and 18 peaks for ARIP-B (Figs. 2 and 3). The MS/MS fragmentation spectra were searched for matching with MASCOT search engine (Matrix Science) and the peptides were identified as shown in Supplementary files 1 and 2. It has been observed that the peptides of ARIP A and ARIP B purified from the seeds of Annona squamosa did not show remarkable match with known RIPs so far reported. The matching was in the range of 1–15% and 1–11% for ARIP-A and ARIP-B (Tables 1 and 2). In the total protein plant description, one uncharacterized protein from Populus trichocarpa showed maximum matches with ARIP-A (% coverage – 2 + 3 + 3 + 5 = 13%) (Table 1) and ARIP-B (% coverage – 2 + 1 + 1 + 3 + 3 = 10%) (Table 2). This plant is reported to have RIP with single ricin B-chain domain, which suggested the possibility of ARIPs could be of Type 2 or single ricin B-chain domain. Interstingly, 13 out of 19 tryptic peptides from ARIP-A and 15 out of 18 tryptic peptides from ARIP-B matched with uncharacterized proteins from various plants reported to have RIP activity. The genome sequence of Annona squamosa genome with the annotated information is not available until now. This could be ascertained as the reason for their low similarity.

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Fig. 2

ESI MS/MS mass spectra of ARIP-A. 19 peaks were observed within the Retention Time of 9 to 17  min.

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Fig. 3

ESI MS/MS mass spectra of ARIP-B. 18 peaks were observed within the Retention Time of 9 to 17  min.

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3.2.2 Sequence alignment

As Annona squamosa is a flowering plant belonging to Magnoliophyta division, the FASTA sequences of RIP proteins from Magnoliophyta families, were collected from the protein database (PDB) as described by Raj and Vennila (2013), matched with LC-MS/ESI-MS data of ARIP-A and ARIP-B (Supplementary files 3 and 4). Nine (9) peptides from ARIP-A showed 100% similarity with a part of amino acid sequence present in RIPs reported from Fabaceae (Abrus precatorius), Viscaceae (Viscum album), Cucurbitaceae (Momordica balsamina, Momordica charantia and Cucurbita moschata), Nyctaginaceae (Bougainvilliea spectabilis) and Phytolaccaceae (Phytolacca americana, Phytolacca dioica and Phytolacca accinosa). However, in ARIP-B, only 7 out of 18 peptides showed 100% similarity with a part of amino acid sequence in RIPs reported in Cucurbitaceae (Momordica balsamina, Momordica charantia and Cucurbita moschata), Phytolaccaceae (Phytolacca americana, Phytolacca dioica and Phytolacca accinosa), Viscaceae (Viscum album), Fabaceae (Abrus precatorius) and Euphorbiaceae (Ricinus communis). The maximum number of peptides identity of ARIP-A was noticed with RIPs reported in Fabaceae and Viscaceae. While ARIP-B showed maximum identity with Phytolaccaceae, Cucurbitaceae and Viscaceae. Most of the RIPs found in these families are reported as Type 2. Hence, we presumed that the ARIPs are likely belong to Type 2. RIP. The presence of carbohydrates during purification steps also substantiates this fact that Type 2B-chain lectin domain have bound to carbohydrates. Although the genome sequence of the Annona squamosa is unavailable for comparison, the peptides of the ARIPs still showed little similarity with part of the amino acid sequence in already known RIPs. This emphasizes that in spite of their difference, all RIPs in Magnoliophyta illustrated a definite motif/pattern in their structure.

All these assumptions led to the conclusion that the ARIP-A and ARIP-B could be the two peptide chains A and B of ricin like Type II ARIP which would be have been denatured and separated as two bands during SDS PAGE.

3.3.1 Antimicrobial activity

The antimicrobial activity against bacterial and fungal strains was found to increase with increase in the concentration of the ARIP (Fig. 4). The ARIP was more effective against bacteria when compared to fungal strains. The antibacterial and growth inhibitory activity of ARIP could be attributed to the presence of lectin (Al-Mamun et al., 2016). Similar antimicrobial activities was also noticed with RIPs reported in Mirabilis expansa (ME1 and ME2) against Pseudomonas aeruginosa (Vivanco et al., 1999). Ricin also have been reported to show antibacterial effect against S. aureus P. aeruginosa, E. coli and E. aerogenes (Al-Mamun et al., 2016). Tobacco ribosomal inactivating protein (TRIP) have also shown strong inhibitory activity against many bacteria (Sharma et al., 2004; Vivanco et al., 1999). Considering the antifungal activities of RIP, Park et al.(2002) have reported that ricin was less toxic to C. albicans ribosomes than saporin. Another study on the RIP protein BE27 from sugar beet by Citores et al (2016) have observed antifungal activity against Penicillium digitatum.

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Fig. 4

Antimicrobial activity of ARIP. ARIP - RIP from the seeds of Annona squamosa. All the experiments were done in triplicates and P < 0.05 was considered statistically significant. Positive controls (C) – Gentamycin and Cyclohexamide were used for bacteria and fungi respectively. ^a denotes comparision of control (C) with ARIP 10 µg/ml; ^b denotes comparision of control (C) with ARIP 20 µg/ml; ^c denotes comparision of control (C) with ARIP 30 µg/ml.

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3.3.2 Antimutagenic activity

Antimutagenic activity helps in cancer prevention. Antimutagenic agents present in plants inhibit the pathogenic effects of mutagenic and carcinogenic substances (Makhafola et al., 2016). With increase in the concentration from 0.5 to 2.0 mg/plate, ARIP exhibited strong antimutagenic activity and inhibition range of of 38.43 to 69.64%. This was found to significantly increase when S9 mix was added (52.71–72.78%) (Table 3). The antimutagenic activity of ARIP was found to be stronger than Viscum album Agglutinin (Hong and Lyu, 2012) but weaker than Ricin (Abbas et al., 2018) and Sechuimin (Wu et al., 1998). All these findings, suggest that ARIP can serve as antimutagenic agents in mutagenesis associated diseases.

3.3.3 Cytotoxic activity

The percentage viability of the AGS Gastric cancer cells treated with different concentrations of ARIP was studied and the IC₅₀ value was observed as 43.02 µg/ml (Fig. 5). The cytotoxic effectiveness was found to be in the order Ricin (IC₅₀ value: 25.57 µg/ml) > ARIP (IC₅₀ value: 43.02 µg/ml) >5 Fluorouracil (IC₅₀ value: 55.34 µg/ml). RIPs have been reported for their potential cytotoxic effect on cancer cells – Riproximin (Ximenia americana) (Voss et al., 2006), Curcin (Jatropha curcas) (Zhang et al., 2017) and Mamorin (Hypsizigus marmoreus) (Pan et al., 2013).

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Fig. 5

Cell growth inhibitory activity of ARIP against AGS Gastric cancer cell lines. ARIP - RIP from the seeds of Annona squamosal. Ricin and 5 Fluorouracil were used as controls for comparison.

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The current study clearly demonstrates the cytotoxic effect of ARIP on cancer cells. These findings suggest that ARIP could be potentially considered as new and novel anticancer compounds for possible drug development against cancer.