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. 2021 Oct 21;26(21):6372.
doi: 10.3390/molecules26216372.

Nano Milk Protein-Mucilage Complexes: Characterization and Anticancer Effect

Ahmed Ali Abd El-Maksoud  1 , Amal I A Makhlouf  2 , Ammar B Altemimi  3 , Ismail H Abd El-Ghany  1 , Amr Nassrallah  4 , Francesco Cacciola  5 , Tarek Gamal Abedelmaksoud  6
Affiliations

Nano Milk Protein-Mucilage Complexes: Characterization and Anticancer Effect

Ahmed Ali Abd El-Maksoud et al. Molecules. .

Abstract

The anticancer activity of natural compounds has recently attracted multidisciplinary research. In this study, the complexation of milk proteins (MP) with Isabgol husk mucilage (IHM) and Ziziphus spina-christi mucilage (NabM) was investigated. In this context, the physicochemical properties of milk protein mucilage complexes (MPMC) including pH, Carr's index, water solubility, and water absorption indices were measured, and the flow behavior was studied. In addition, the amino acid profile, protein digestibility, and phenolic and flavonoids content of MPMC were explored, and the microstructure of the complexes was visualized using transmission electron microscopy. The antioxidant and anticancer potencies of MPMC against two cancerous cell lines, human liver cancer HEPG-2 and breast cancer MCF-7, in comparison with two normal cell lines, namely, Bj-1 and MCF-12F, were tested using neutral red uptake assay. The results revealed that MPMC had scavenging activity against DPPH, ABTS, and HS radicals. Moreover, MPMC has the potential to prevent DNA damage induced by oxidative stress in Type-Fenton's reaction. The results of the neutral red assay showed significant growth inhibition of both HEPG-2, MCF-7, whereas no significant cytotoxic effect was detected against Bj-1 and MCF-12F. RT-qPCR results indicated MPMC stimulated apoptosis as revealed by the upregulation of the pro-apoptosis gene markers Casepase-3, p53, Bax. Meanwhile, the anti-apoptosis Bcl-2 gene was downregulated. However, no significant difference was observed in normal cell lines treated with MPMC. In conclusion, MPMC can be considered as a promising anticancer entity that can be used in the development of novel cancer therapeutics with comparable activity and minimal side effects compared to conventional cancer chemotherapies.

Keywords: Isabgol husk mucilage; Nabeq mucilage; anticancer activity; milk proteins; milk proteins mucilage complexes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Fourier-transform infrared spectra of MP, IHM, and MP/PHM. (B) Fourier-transform infrared spectra of MP, IHM, and MP/NabM. NabM: Nabeq mucilage, IHM; Isabgol husk mucilage, MP: milk proteins concentrate, MP/IHM: milk proteins/Isabgol husk mucilage complex, MP/NabM: milk proteins/Nabeq mucilage complex.
Figure 1
Figure 1
(A) Fourier-transform infrared spectra of MP, IHM, and MP/PHM. (B) Fourier-transform infrared spectra of MP, IHM, and MP/NabM. NabM: Nabeq mucilage, IHM; Isabgol husk mucilage, MP: milk proteins concentrate, MP/IHM: milk proteins/Isabgol husk mucilage complex, MP/NabM: milk proteins/Nabeq mucilage complex.
Figure 2
Figure 2
HPLC analysis of polyphenolic profiles of (A) IHM and (B) NabM.
Figure 3
Figure 3
Total phenolic and total flavonoid content (A,B) and antioxidant activity (C–E) of MP, IHM, NabM, and MPMC. MP: milk proteins concentrate; IHM: Isabgol husk mucilage; NabM: Nabeq mucilage; TPC: total phenolic content; GAE: gallic acid equivalent; TF: total flavonoid; CE: catechin equivalent; DW: dry wight; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid; HS: hydroxyl scavenging; Values are means ± SD (n = 3). Measurements with different letters (a, b, c, d, and e) are significantly different (p < 0.05).
Figure 4
Figure 4
Amino acid profile of MP, MP/IHM, and MP/NabM. MP: milk proteins concentrate, MP/IHM: milk proteins/Isabgol husk mucilage complex and MP/NabM: milk proteins/Nabeq mucilage complex.* essential amino acids.
Figure 5
Figure 5
Transmission electron microscopy of (A) MP; (B) IHM; (C) NabM; (D) MP/IHM; and (E) MP/NabM. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
Figure 5
Figure 5
Transmission electron microscopy of (A) MP; (B) IHM; (C) NabM; (D) MP/IHM; and (E) MP/NabM. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
Figure 5
Figure 5
Transmission electron microscopy of (A) MP; (B) IHM; (C) NabM; (D) MP/IHM; and (E) MP/NabM. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
Figure 6
Figure 6
The anticancer activity of MP, MP/NabM, and MP/IHM on HepG-2 (A), MCF-7 (B), Bj-1 (C), and MCF-12F (D) cell lines at different concentrations (1, 5, 10, and 20). The cytotoxicity was evaluated calorimetrically by neutral red uptake assay. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex. Three replicates of each treatment were analyzed. Measurements with different letters (a, b, c and d) are significantly different (p < 0.05).
Figure 7
Figure 7
Morphology of human cancer cell lines and normal cell lines; HepG-2 (A), MCF-7 (B), MCF12F (C), and BJ-1 (D) before and after treatment with MP and MPMC. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
Figure 8
Figure 8
Protein levels of apoptosis biomarker. The level of Caspase-3 (A), p53 (B), Bax (C), and Bcl-2 (D) in HepG-2, MCF-7, Bj-1, and MCF-12F treated with or without the IC50 of MP, MP/NabM, and MP/IHM. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex. Data are average of triplicates. Measurements with different letters (a, b, c and d) are significantly different (p < 0.05).
Figure 9
Figure 9
DNA damage protection by MP/NabM and MP/IHM. (A) The protective capacity MP/NabM, MP/IHM, and MP against RNH1 plasmid DNA damage induced by Fenton’s reagent. (B) The protective capacity MP/NabM, MP/IHM, and MP against genomic DNA damage. Lane 1: DNA control, lane 2: DNA treated with Fenton’s reagent, lane 3: DNA treated with Fenton’s reagent plus MP/NabM, lane 4: DNA treated with Fenton’s reagent plus MP/IHM, and lane 5: DNA treated with Fenton’s reagent plus MP. MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
Figure 10
Figure 10
Analysis of apoptosis genes transcript in HepG-2, MCF-7, Bj-1, and MCF-12F cells treated with MP, MP/IHM, and MP/NabM. Profiling of mRNA transcript levels of key pro- (Casp3 (A), p53 (B), Bax (C) and anti-apoptotic Bcl-2 (D)). Gene expression levels were quantified after 72 h by RT-qPCR employing 18S as a housekeeping gene for normalization as detailed in the methods. Significant differences between the means of individual treatments and control were analyzed by one-side Student’s t-test. Histograms represent mean expression level as fold change SD for 3 technical and 2 biological replicas with different letters (a, b and c) are significantly different (p-value ≤ 0.05). MP: milk proteins concentrate; MP/NabM: milk proteins/Nabeq mucilage complex; MP/IHM: milk proteins/Isabgol husk mucilage complex.
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