Introduction

Milk is the primary source of nutrients for young mammalians. Lactoferrin (Lf) is an 80-kDa iron-binding glycoprotein of the transferrin family, which was first fractionated as an unknown ‘‘red fraction’’ from cows’ milk by Sorensen. It is recognized as being nutritionally balanced and has therefore attracted a lot of scientific interest over the years. Various properties of intact milk proteins have been reported including satiating, antimicrobial, mineral binding, antilipidaemic and anticancer properties (Anderson and Moore, 2004; Chatterton et al., 2006; Clare and Swaisgood, 2000; Cross et al., 2007; Nakamura et al., 2013). Identification of large number of peptides in milk protein hydrolysate makes the milk proteins as one of the most important source of bioactive peptide. Several studies have suggested that milk protein-derived bio active peptides (BAPs) may be used as preventative/prophylactic agents to alleviate symptoms of various diseases in humans. Side-effects of various drugs used to cure/slow down the progress of specific diseases in humans may sometimes outweigh their benefits (Li-Chan, 2015; Saadi et al., 2015). Further increasing awareness regarding potential benefits of milk protein derived bioactive peptide among the people, laid path of growing milk nutraceutical market (Nagpal et al., 2011). Lf is a non-heme iron binding glycoprotein with molecular weight of 78 kDa that contains around 690 amino acid residues. It belongs to the transferrin (Tf) family. It is one of the major proteins of all exocrine secretions including saliva, tears, semen, vaginal fluids, gastrointestinal fluids, nasal mucosa and bronchial mucosa of human being (Iigo et al., 2009; Birgens et al., 1985). Lf is also found in milk of bovine, caprine, camel and humen (Baker and Baker, 2005). Lf is also known for its anti-bacterial, antifungal, antiviral, antimicrobial, anti-oxidant, anti-inflammatory, anti-parasitic, anti-allergic and most importantly anticancerous properties (Iigo et al., 2009; Parhi et al., 2012). Lf is the second most abundant milk protein after casein and its highest concentration is found in human colostrums and then human milk followed by cow milk (Sanchez et al., 1992). Development of drug-resistant cancers imposed question mark on the use of chemotherapeutic agents. This limitation raise the need of a natural substitute that has generalized acceptance and can possibly completely eradicate the primary tumor, thus eliminating the risk of recurrence. In this context Lf has got the potential to be used as anticancer bio-molecule. This review represents the properties and structure of Lf along with the function and therapeutic importance.

Lactoferrin: Structure

The structure of Lf consists of a single polypeptide chain which is folded into two lobes (N and C lobes) with 33–41% homology (González-Chávez et al., 2009). Both lobes are linked by an α-helical residue, making Lf a flexible molecule (Fig. 1). The two lobes of Lf are made of α-helix and β-sheet, and each lobe can bind either Fe⁺² or Fe⁺³ ions in synergy with the carbonate ion (CO₃^2–) (Iafisco et al., 2011). Amongst transferrin family the lactoferrin has highest iron binding affinity. Lf is the major iron transporter protein in blood plasma (Iafisco et al., 2011). In its natural form lactoferrin, is partially saturated with iron and hence can be fully saturated with iron from the external environment (Kanwar et al., 2008; Tsuda et al., 2004). Lf acts as a signaling molecule in various pathways to exert their cytotoxic effects. Human Lf (hLf) and bovine Lf (bLf) cause cell cycle arrest and leads to apoptosis (programmed cell death) in cancer cells while bovine lactoferriin (bLf) inhibits cell growth by triggering mitochondrial related apoptosis (intrinsic apoptotic pathway) and disrupting the cell membrane.

Fig. 1: Three dimensional folding of buffalo lactoferrin (Sources: Karthikeyan et al., 1999)

Sources of Lactoferrin

Lf is an important part of the innate immune system (Wakabayashi et al., 2006). Lf is continuously synthesized in body and is released into the exocrine fluids like saliva (Reitamo et al., 1980), tears (McClellan, 1997) and vaginal fluids (Valore et al., 2002), or only at well-defined stages of cell differentiation such as granules of neutrophils (Breton-Gorius et al., 1980). Glandular epithelial cells secrete Lf in milk source. Milk is by far the most abundant source of lactoferrin with human colostrum, the early milk, containing up to 7 g/l (Masson and Heremans, 1971). There is a great variation in the concentration of lactoferrin in other human body fluids. The concentration in tears is as high as 2 mg/ml whereas that in blood is normally only as high as 1 μg/ml, although it can rise as high as 200 μg/ml in the inflammatory situation (Masson and Heremans, 1971). The mean lactoferrin (Lf) concentration determined by electroimmunodiffusion (EID) assay of whey preparations from 80 quarters of 20 normal lactating cows was 0.35 mg/ml (Harmon et al., 1975). Although lactoferrin is found in the milk of most mammals its concentration is quite variable and dependent on the stage of lactation. During an infection or an inflammatory condition, the levels of Lf are raised in the body (Caccavo et al., 2003) making Lf a biomarker for inflammatory conditions.

Lactoferrin market is segmented on the basis of application which includes food products, infant formula, sports and functional food, pharmaceuticals, feed products and personal care products. Among these entire segments pharmaceuticals segment is expected to account for the major share in terms of revenue contribution followed by cosmetic segment during the forecast period. Various functional properties such as iron bioavailability, anti carcinogenic effect, antibacterial effect, anti-oxidant effect and others support in making or formation of varieties of drugs which is supporting the growth of lactoferrin market in pharmaceutical segment.

Fig. 2: Functions of lactoferrin

Functions of Lactoferrin

Risk of development of resistance to antibiotics raises the needs for alternatives antimicrobials and lactoferrin is one of the promising antimicrobial molecule that have potential to fills the gap (Li et al., 1995). The antibacterial activity of lactoferrin is mediated through its iron sequestering ability by virtue of which iron become inaccessible to bacteria and hampered their growth and division (Bullen et al., 1972).  Lf and Lf derived peptide has bacteriostatic activity against both Gram positive and Gram negative bacteria (Ellison et al., 1988). Staphylococcus epidermidis is one of the most predominant infectious agents in individual implemented with intraocular lenses leading to a characteristic biofilm formation on the soft contact lenses. It is observed that Lf increases the sensitivity of this bacterium by binding to the anionic cell wall preferentially to vancomycin thereby allowing its entry into the bacteria (Leitch and Willcox, 1999b). Lf facilitate the penetration of lysozyme as it binds to teichoic acid and compensate the charges on cell wall (Leitch and Willcox, 1999a). Lf causes depolarization of the bacterial membrane leading to membrane penetration and eventually metabolic injury. Lf is also used to treat periodontal diseases by acting against plaque forming oral microorganisms like Streptococcus mitis, Streptococcus gordoni, Streptococcus salivarius and Streptococcus mutans.

Antimicrobial Effect of Lactoferrin on Gram-Negative and Gram-Positive Bacteria

It is more effective against gram positive bacteria as found in the following research. All the species of Bacillus cereus, Staphylococcus epidermidis, Campylobacter jejuni and Salmonella were cultured on agar plate with and without Lf (Jahani et al., 2015).

Lf also prevents the colonization of Giardia lambia, a most common protozoal infection of human intestine by acting on Giardia trophozoites plasmalemma, endomembrane and cytoskeleton (Ochoa et al., 2008). In another study, antibacterial activity of bLf hydrolysate by using different enzyme including, rennet and pepsin were assessed against Escherichia coli and Bacillus subtilis. The study revealed that Lf-cin B was the most potent antibacterial peptide and was isolated from both rennet and pepsin LFH (Elbarbary et al., 2010). It was demonstrated that pepsin hydrolysate derivatives of bLf had stronger bifidogenic activity than natural against Bifidobacterium breve and Bifidobacterium longum species (Oda et al., 2013). Several modifications have been attempted in bLf in order to use it as a food preservative. It was found that glycosylated lactoferrin (gLf) showed substantial Fe-binding capacity and excellent emulsifying properties and also revealed its ability to inhibit the growth of E. coli at 50°C completely (Nakamura, 2002). Hence, these findings offer new possibilities for Lf as a food preservative. In another study it was observed that nano-formulated Fe-bLf was more effective in the treatment of Salmonella-infected mice than the standard therapy using ciprofloxacin (Gupta et al., 2014).  Lf has ability to damage fungal cell membrane that alters its permeability and also its iron chelating properties attributed to antifungal activity (Wakabayashi et al., 2000). Lf also exhibits antiprotozoal activity but the mechanism varies from its antibacterial and antifungal aspects. Studies proved that although Lf had no role in inhibiting the entry of these parasites into the system but did not allow the growth of these protozoans in the host (Cintra et al., 1986).

Use of lactoferrin as antiviral compound is one of the most recent properties. Although the research regarding antiviral activity of Lf is in early phase, however, there are only a very few cases in which Lf failed to benefit as an antiviral activity. Lf exhibited antiviral activity against a number of viruses including herpes simplex virus, cytomegalovirus, hepatitis B and C virus (HBV and HCV) and human immunodeficiency virus (HIV) (Hara et al., 2002; Ikeda et al., 1998; Harmsen et al., 1995; Roy et al., 2012). A new perspective in the studies of antimicrobial activity of Lf is due to its potent prophylactic and therapeutic ability in a broad spectrum. Unlike to all these antimicrobial effects, in some protozoans like trichomonas, Lf helps in effective binding, and successful internalization in these parasites (Tachezy et al., 1998).

Table 1: Source, action and functional role of lactoferrins

Following research shows that Lf is effective in coliform mastitis-

• The mean concentration of Lf in the milk of cows with subclinical mastitis (0.2-1.2 mg/ml) has been shown to be higher than in milk of normal cows (Hagiwara et al., 2003).
• High concentration of Lf promotes phagocytic activity in the mammary gland and the activation of bovine complement (Kai et al., 2002).

Lactoferrin and Immunity

Beside diverse function of Lf in various body fluids, iron free form of Lf is the integral component of cytoplasmic secondary granules of neutrophils thus have role in first line defense (Fig. 3). During inflammation, Lf is released and the concentration of Lf at the site of inflammation is increased from 0.4–2.0 μg/ mL to 200 μg/ mL playing a major role in the feedback mechanism of inflammatory response (Farnaud and Evans, 2003). In the kidney Lf is synthesized locally where, it sequestrates free iron from urine and makes it available for metabolic functions (Abrink et al., 2000).

Lf acts as immune modulator by interacting with specific cell receptors of epithelial and immune cells and as a lipopolysaccharide to pro-inflammatory bacterial elements (Na et al., 2004; Elass-Rochard et al., 1995; Legrand et al., 2008). At cellular level Lf significantly affects the differentiation, maturation, activation, migration, proliferation and functions of immune cells by using, nuclear factor-kappa B (NF-κB) and MAP kinase signaling pathway (Gahr et al., 1991). Lf from bovine milk showed proteinase inhibitory activity against Porphyromonas gingivalis, a bacterial pathogen, by inhibiting Arg and Lys-specific proteolytic activities (Manzoni et al., 2012). The bovine Lf at molecular level influence maturation of lymphocyte and release of cytokines in bone marrow microenvironment (Touyz et al., 2000). Anti-inflammatory action of Lf alleviates stress by preventing the excess inflammatory response (Ye et al., 2014). It was demonstrated that Lf knockout mice shown high susceptibility to inflammation-induced colorectal dysplasia, mainly due to NF-κB and AKT/mTOR signaling, regulation of cell apoptosis and proliferation. On the basis of above study it can be inferred that anti-carcinogenic property of Lf is attribution of its anti-inflammatory function (Gutteridge et al., 1979).

Free form of iron plays a pivotal role in generation of reactive oxygen species (ROS) and leads to lipid peroxidation of cell membranes using iron-dependent Haber-Weiss reaction. Inefficiency of certain vital enzyme like, catalase, glutathione peroxidase and superoxide dismutase leads to over production of hydroxyl radicals further increases the oxidative stress (Raetz et al., 1991). It is hypothesized that iron sequestration by Lf from the microenvironment limits the oxidative damage to bio-membranes by hampering lipid peroxidation. Lf also regulates the systemic inflammatory response in controlled manner so that there is minimum damage to surrounding tissues (Gahr et al., 1991; Pajkrt et al., 1996). Antioxidant mechanism is one of the attribute by virtue of which oral administration of Lf shown to support improved immune response (Mulder et al., 2008).

Lf is considered as important component in first line host defense, as it play vital role in innate as well as adaptive immune response (Legrand et al., 2008 and Kruzel et al., 2002). It was revealed that Lf potentiate the phagocytic activity of neutrophils (Wakabayashi et al., 2003), increased activity of NK  and also involved in macrophages activation by increased production of cytokines and nitric oxide (NO), reduces the proliferation of intracellular pathogens (Kawai et al., 2007). Cytokines TNF-α, IL-6 and IL-1β by Lf according to the requirement helps to confer its immune modulatory activity. Lf regulate the production of antigen presenting cells (APCs) like, macrophages, dendritic cells and B cells which presents the processed antigen to CD4+ T cells via major histocompatibility complex II (MHC II) (Puddu et al., 2009), thereby it play active role in specific immune response against pathogens. Lf found to reduces the production of cytokines, TNF-α, IL-6 and IL-1β that were induced by Bacille-Calmette-Guerin strain of Mycobacterium bovis.

Anticarcinogenic Activity

It is reported that all T cell subsets including δγ T cells have been expressed Lf receptors (Mincheva-Nilsson et al., 1997). Lf has shown to up regulate the leukocyte function associated antigen (LFA-1) which is an adhesion molecule present on CD4+ and CD8+ T cells, in human peripheral blood mononuclear cells when cultured in presence of human Lf (Zimecki et al., 1999).  Expression of human T cell ζ–chain, T cell receptor complex involved in receptor signaling were enhanced by hLf (Sfeir et al., 2004) observed that when concanavalin A (ConA) activated murine splenocytes were cultured in the presence of bovine or human Lf resulted in reduced production of IFN-γ and IL-2. There are various studies that proven the immune modulatory function of Lf such as oral delivery of Lf to the mice bearing tumor cells showed an increase in lymphoid and intestinal CD4+ and CD8+ T cells (Wang et al., 2000); increased population of circulating leukocytes and granulocyte were seen in mice with orally administered Lf (Wakabayashi et al., 2006). Recently presence of lactoferrin in feces has been introduced as a biomarker for the diagnosis and monitoring of inflammatory bowel disease (IBD). It could also be used as tool to investigate and quantify the effect of granulocyte and monocyte adsorptive apheresis (GMA) in ulcerative colitis (UC) (Hashiguchi et al., 2015). Hence, lactoferrin has diverse role, it ranges from immunodiagnostic tool to immunotherapeutic agent.

Effect of bovine lactoferrin (bLf) on growth of breast cancer cells by zhang et al. (2015), three independent experiments were done on breast cell line, shows that bLf induced Apoptosis.

Fig. 4: Role of lactoferrin in the activation of immune cells

Lactoferrin enters in the intestinal microvilli through the help of lactoferrin receptors and transferrin receptors present on the mucosal surface of the intestinal cells. The lactoferrin molecule further boosts up the immune response due to IFN-γ, TNF-α, IL-6 and by activating NK cells, PMNs and CD3+ and CD4+ T cells.

Finally the lactoferrin enters the cells by receptor mediated endocytosis where it is released within the cells once the receptors are digested by endosomes. The lactoferrin induces release of cytochrome C from mitochondria which further activates caspase 3 to cause apoptosis in tumour cells.

Conclusions

The advantages of this natural molecule prove its potential as a natural therapeutic agent that can be used in various fields of research including cancer. The role of Lf as anti-bacterial and anti-fungal agent had been beneficial in its use as a bactericidal and fungicidal agent in lotions and creams. Its use can be extended to topical applications as well. An interesting aspect of using Lf as an anti-cancer agent by delivering it to the body in the form of ice-creams, tablets and oral supplements in the form of NPs have been researched upon. With its role in being able to combat deadly viruses like HCV and HBV also poses a need for its use as an anti-viral agent for human immunodeficiency virus (HIV) and other potent viruses that cause health risks. The role of this natural molecule as anti-inflammatory agent needs further research. It stands as a biomarker for inflammatory conditions and its potential role as a therapeutic molecule needs to be taken forward.

1. Initial research has shown its potential as a novel nutraceutical for enhancing animal production, health and welfare.
2. However research on this topic is largely focused on lab animals, how Lf enhances antioxidant status, immunity and performance needs to be ascertain in livestock and poultry.
3. The role of supplementary Lf in prevention and management of mastitis and anemia needs special attention.

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