Camel Α-lactalbumin Research Articles

Therapeutic efficacy of α-lactalbumin coated oleic acid based liposomes against colorectal carcinoma in Caco-2 cells and DMH-treated albino rats

Colorectal cancer (CRC) is a malignant tumor recognized as a major cause of morbidity and mortality throughout the world. Therefore, novel liposomes of oleic acid coated with camel α-lactalbumin (α-LA coated liposomes) were developed for their potential antitumor activity against CRC, both in vitro and in DMH-induced CRC-modeled animal. In vitro results indicated the high safety of α-LA coated liposomes towards normal human cells with potent antitumor activity against Caco-2 cells at an IC50 value of 57.01 ± 3.55 µM with selectivity index of 6.92 ± 0.48. This antitumor activity has been attributed to induction of the apoptotic mechanism, as demonstrated by nuclear condensation and arrest of Caco-2 cells in sub-G1 populations. α-LA coated liposomes also revealed a significant up-regulation of the p53 gene combined with a down-regulation of the Bcl2 gene. Moreover, in vivo results revealed that treatment of induced-CRC modeled animals with α-LA coated liposomes for six weeks markedly improved the CRC-disorders; this was achieved from the significant reduction in the values of AFP, CEA, CA19.9, TNF-α, IL-1β, MDA, and NO coupled with remarkable rise in SOD, GPx, GSH, CAT, and CD4+ levels. The histopathological findings asserted the therapeutic potential of α-LA coated liposomes in the treatment of CRC. Therefore, the present results proved the antitumor activity of α-LA coated liposomes against CRC through the restoration of impaired oxidative stress, improved immune response, and reduced inflammation. Communicated by Ramaswamy H. Sarma

SIRT1 Mediates Neuroprotective and Neurorescue Effects of Camel α-Lactalbumin and Oleic Acid Complex on Rotenone-Induced Parkinson's Disease.

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder. Presently used therapies for PD have severe side effects and are limited to only temporary improvement. Therefore, a new therapeutic approach to treat PD urgently needs to be developed. α-Lactalbumin, the most abundant milk protein in camel milk, has been attributed to various medicinal properties. This study intended to investigate the neuroprotective efficacy of the camel α-lactalbumin and oleic acid (CLOA) complex. One mechanism postulated to underlie neuroprotection by the CLOA complex is the induction of silent information regulatory protein (SIRT1). SIRT1 is known to be involved in several pathological and physiological processes, and it has been suggested that SIRT1 plays a protective role in PD. Oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis are involved in PD pathogenesis. Our results revealed that SIRT1 inhibits oxidative stress by maintaining HIF-1α in a deacetylated state. SIRT1 upregulates the expression of FOXO3a and HSF-1, thus inhibiting apoptosis and maintaining the homeostasis of cellular proteins. Increased SIRT1 expression reduces the levels of TNF-α, IL-6, and IL-8, which in turn inhibits neuroinflammation. In addition to SIRT1, the CLOA complex also enhances the expression of survivin and leptin and promotes the survival of neuroblastoma cells. Altogether, our results suggest that the CLOA complex might be a novel therapeutic molecule that could ameliorate neuronal cell damage in PD.

Elucidating the Neuroprotective Role of Formulated Camel α-Lactalbumin-Oleic Acid Complex by Curating the SIRT1 Pathway in Parkinson's Disease Model.

Parkinson's Disease (PD) is characterized by increased oxidative stress and decreased level of dopamine. At present, the therapeutic interventions of PD are associated with undesirable adverse effects. To overcome these side effects, a new candidate bioinspired molecule is needed for the management of PD. Camel α-lactalbumin (α-LA) is the most abundant protein in camel's milk and has a potential to act as a nutraceutical supplement for neurological functions. Oleic acid, a monounsaturated fatty acid, has been widely associated with a reduced risk of PD. The present study aimed to formulate the camel α-LA and oleic acid (CLOA) complex under specific conditions and to evaluate its efficacy as a neuroprotective in rotenone induced PC12 cell model of PD. Our results demonstrated that removal of Ca++ ions from camel α-LA by EDTA enhances its binding efficiency with oleic acid, and the complex was characterized by UV-CD, ANS fluorescence spectroscopy, and NMR spectroscopy. Moreover, CLOA complex treatment reduced the oxidative stress and increased the cell viability by enhancing the level of dopamine and the expression of SIRT1, FOXO3a, HIF-1α, and HSF-1. We also validated the neuroprotective role of the complex by incubating the cells with CLOA complex prior to rotenone treatment. We inferred from the outcome of the results that the individual entity, i.e., α-LA or OA, is not as effective as the complex. Taken together, our study indicates that CLOA complex might be a potential candidate for the development of future therapeutic drugs for PD.

Effect of nonenzymatic deamidation on the structure stability of Camelus dromedarius α-lactalbumin

Camelid α-lactalbumin is the only known protein that can undergo nonenzymatic deamidation on two Asn residues. This leads to the generation of a mixture of unusual isoAsp and d-Asp residues that may impact health. The effect of deamidation on camel α-lactalbumin instability was investigated. Circular dichroism showed that the altered protein acquired secondary structure resulting in an increase in α-helix content. In good agreement, the 3D structure of camel α-lactalbumin determined by X-ray crystallography, displayed a short additional α-helix probably induced by deamidation, compared to the human and bovine counterparts. This α-helix was located in the C-terminal region and included residues 101–106. Differential scanning calorimetry together with the susceptibility to thermolysin showed that the deamidation process reinforced the structural stability of the α-lactalbumin at high temperature and its resistance toward proteolysis.

The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk

The effect of pH (4.3 or 6.5) and heat treatment (70°C or 90°C for 30min) on the foaming and interfacial properties of α-lactalbumin extracted from camel milk were studied. The increased temperature treatment changed the foaming properties of camel α-lactalbumin solution and its ability to unfold at the air–water interface. At neutral pH, heat treatment was found to improve foamability, whereas at acid pH (4.3) this property decreased. Foams were more stable after a heat treatment at pH 4.3 than at 6.5, due to higher levels of protein aggregation at low pH.Heat treatment at 90°C for 30min affected the physicochemical properties of the camel α-lactalbumin by increasing free thiol group concentration at pH 6.5. Heat treatment also caused changes in α-lactalbumin’s surface charge. These results also confirm the pronounced aggregation of heated camel α-lactalbumin solution at acid pH.

Proteomic profiling of camel and cow milk proteins under heat treatment

Cow and camel milk proteins before and after heat treatment at 80°C for 60min were identified using LC/MS and LC–MS/MS following monodimensional electrophoresis. The database used for the identification of camel and cow proteins was set from http://www.uniprot.org/. The obtained results showed that, after heating, camel milk at 80°C for 60min, camel α-lactalbumin (α-la) and peptidoglycan recognition protein (PGRP) were not detected while camel serum albumin (CSA) was significantly diminished. When heating cow milk at 80°C for 60min, α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were not significantly detected. Moreover, 19 protein bands from SDS-PAGE were analyzed and a total of 45 different proteins were identified by LC–MS/MS. Casein fractions were kept intact under a heat treatment of 80°C during 60min of both camel and cow milks. Camel and bovine whey proteins were affected by a heat treatment of 80°C for 60min.

Structure and Stability Analysis of Cytotoxic Complex of Camel α-Lactalbumin and Unsaturated Fatty Acids Produced at High Temperature

α-Lactalbumin (α-La), together with oleic acid can be converted to a complex, which kills tumor cells selectively. Cytotoxic α-La -oleic acid and a-La -linoleic acid complexes were generated by adding fatty acid to camel holo a-La at 60°C (referred to as La-OA-60 and La-LA-60 state, respectively). Structural properties of these complexes were studied and compared to the camel α-La. The experimental results show that linoleic acid induces a-La partial unfolding but oleic acid does not change the protein structure significantly. Also the stability of La-OA-60 and La- LA-60 toward thermal denaturation was measured. The order of temperature at the transition midpoint is as follows: La-LA-60 < La-0A-60 < α-La. La-0A-60 complex inhibited tubulin polymerization in vitro. Although the structures of La-0A-60 and La-LA-60 were different, these two complexes had similar cytotoxic effect to DU145 human prostate cancer cells. Samples of La-0A-60 that have been renatured after denaturation lost the specific biological activity toward tumor cells.

Comparative study on heat stability of camel and bovine apo and holo α-lactalbumin

The stability of camel alpha-lactalbumin (alpha-la) against heat denaturation was measured, using circular dichroism (CD) and fluorescence spectroscopy, as well as differential scanning calorimetry (DSC). The experiments were performed in the presence of saturating concentrations of calcium as well as in the presence of EDTA, yielding to the apo form of alpha-la. The change in heat capacity (DeltaCp) suggests a greater contribution of hydrophobic interactions to the stability of holo camel alpha-la than in its bovine counterpart. Overall the results obtained in this study suggest a greater stability of camel alpha-la than the bovine protein in both holo and apo states. Also CD experiments showed similar secondary structure for camel and bovine alpha-la and secondary structure of camel alpha-la was better preserved than that of bovine alpha-la during heat denaturation. The differences in thermal stability between the proteins from two species can be primarily ascribed to the difference in the quantity of hydrophobic interactions involved in their folding.

Enzymatic digestion and antioxidant activity of the native and molten globule states of camel α-lactalbumin: Possible significance for use in infant formula

Native- and molten globule states of α-lactalbumin (α-La) from camel and bovine milk were used for comparative assessment of digestibility and antioxidant activity. The proteolysis assessments were performed in the presence of gastrointestinal enzymes, using the o-phthaldialdehyde assay, and the antioxidant activity was carried out using a 2,20-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid based method. Camel and bovine α-La revealed similar sensitivity to proteolysis by pepsin. The degree of hydrolysis (DH) of camel α-La by either trypsin or chymotrypsin was noticeably higher than that of the bovine protein counterpart. This can be explained by the different conformational and structural features of these proteins, as shown by studies of intrinsic- and 8-anilinonaphthalene-1-sulfonic acid fluorescence. The greater antioxidant activity of camel α-La could be explained by the higher content of antioxidant amino acid residues and different conformational features between bovine and camel α-La. The results may suggest that α-La produced from camel milk may be used for infant formulae as an alternative to that produced from bovine milk.