**3.2 Camel milk lactoferrin as an antimicrobial, anticancer, and immunomodulatory agent**

Lactoferrin is a highly conserved molecule. It possesses high degree of sequence homology and exerts multiple identical functions across mammalian species. Its


#### **Table 2.**

*Applications of lactoferrin.*

ability to act as an antibacterial, antifungal, antiviral and antiparasitic, anti-inflammatory and immunomodulatory agent is shared amongst most mammalian species and has already been discussed [56–58]. More specifically, it inhibits growth of *Escherichia coli, Klebsiella pneumonia, Clostridium, Helicobacter pylori, Staphylococcus aureus, Candida albican*s, etc.

According to studies, the most therapeutic effects of camel milk are due to lactoferrin and immunoglobulins. Redwan & Tabll, 2007 reported that lactoferrin of camel milk has anti-viral activity and inhibits the virus entry into the cells. The camel milk lactoferrin stops HCV entry and replication in infected HepG2 cells two times higher than lactoferrin in human, bovine, and sheep milk. Generally, camel milk lactoferrin may directly interact with viral molecules or receptors (heparan sulfate) on the cell surface and prevent the virus's attachment to the host cells and thus hinder infection. The virucidal mechanism of camel milk lactoferrin depends on its alpha-helical structure and cationic nature [59]. The antiviral effects of lactoferrin from camel milk have been demonstrated against many viruses. The mode of action behind this activity is the neutralization of virus particles and inhibition of their replication. Camel milk lactoferrin also has anti-pathogenic activity against human immunodeficiency virus, hepatitis B and C, cytomegalovirus as well as herpes simplex virus-1 infection. Not only this, but camel lactoferrin's immunomodulatory role is exemplified by the fact that it modulates the activation and maturation of various immune cells such as neutrophils, macrophages, and lymphocytes [60].

#### *Medicinal Potential of Camel Milk Lactoferrin DOI: http://dx.doi.org/10.5772/intechopen.108316*

An earlier study on camel milk lactoferrin has demonstrated the ability to inhibit the growth of colon cancer cells line HCT-116. Camel milk lactoferrin exerted antioxidant activity through scavenging NO and the DPPH free radical. It has shown the capability to furnish reducing power as evident by total antioxidant assays. Camel milk lactoferrin also inhibited DNA damage most likely through binding catalytic iron [5].

Camel milk lactoferrin exhibits an anti-inflammatory activity against IL-1β induced activation of osteoarthritis associated chondrocytes in humans by blocking the NFkappa B mediated signaling. Furthermore it inhibited cyclooxygenase-2 expression and PGE2 production in stimulated osteoarthritis chondrocytes. N. Rasheed et al., 2016 have reported that camel lactoferrin has cartilage protective and anti-arthritic activity. This novel mode of action of camel milk lactoferrin is very important in understanding the mechanisms behind its anti-inflammatory or anti-arthritic effects [61]. The above studies on lactoferrin derived from camel milk highlight the clinical relevance.

#### **3.3 Anticancer potential of lactoferrin from other mammalian species**

Human and Bovine lactoferrin has been suggested to be able to act in tumor prevention and treatment [62, 63]. The lactoferrin preventive effect has been demonstrated in several animal models bearing different types of malignancies, including lung, tongue, esophagus, liver, and colorectal tumors [64–67]. Whereas lactoferrin treatment, was found to be effective in inhibiting growth, metastasis, and tumorassociated angiogenesis [63, 68, 69].

Bovine lactoferrin prevents development of chemically induced tumors. This effect has been confirmed in studies conducted on laboratory rodents. Based on in vivo studies, oral administration of lactoferrin to rodents significantly decreased the chemically induced carcinogenesis in various organs such as breast, esophagus, tongue, lung, liver, colon, and bladder. It also hindered angiogenesis and decreased the incidence of metastases in experimental mice [67].

Furthermore, the combined administration of Lactoferrin and temozolomide enhances the effect of chemotherapy both in vitro and in vivo [55] Similarly, humans suffering from lung cancer undergoing chemotherapy had increased immune system response after taking human lactoferrin post-treatment [70].

Lactoferrin from a bovine source is a promising candidate as an anticancer agent [71] Although bovine milk contains lactoferrin, the human form has been found to be far more potent. Animal studies with mice or rats have shown beneficial effects of bovine lactoferrin ingestion as it can inhibit carcinogen–induced tumors in the colon, esophagus, lung, tongue, bladder, and liver [72].

The anticancer effect of lactoferrin has been extensively studied, and it has been observed that in the presence of Lactoferrin, cancer cells suffer significant damage. It is known to cause cell cycle arrest, damage to the cytoskeleton, and induction of apoptosis, in addition to decreasing cell migration [63, 73]. It decreased the viability and growth of breast cancer cell lines (HS578T and T47D). It also stopped cancer cell growth during the cell cycle and disrupted the cancer cell membrane [74]. Bovine lactoferrin efficiently inhibited the growth of breast cancer cells, suggesting that it has a potential to act as an anti-cancer agent against breast cancer [63, 75].

Lactoferrin helps to prevent the growth of cancer cells and shrinks the cancer cells. It is also known for its inhibitory action on cancer cell proliferation and its anti-inflammatory as well as antioxidant abilities against them [75]. Lactoferrin

expression levels are decreased in colorectal cancer as compared with normal tissue. Lactoferrin knockout mice demonstrated a great susceptibility to inflammationinduced colorectal dysplasia. Treatment of knockout mice with lactoferrin postchemotherapy accelerated the reconstitution of the immune system, reducing the chances for infection, following chemotherapy treatment. Additionally, lactoferrin is significantly downregulated in specimens of nasopharyngeal carcinoma (NPC) and is negatively associated with tumor progression, metastasis, and prognosis of patients with NPC [76].

Lactoferrin was shown to have preventive effects against gastrointestinal cancers, such as cancer of the colon, stomach, liver, and pancreas, and against metastasis of such neoplasms [77, 78]. Xu *et al*. (2010) demonstrated that bovine lactoferrin induces apoptosis in stomach cancer, thereby suppressing it [79]. Oral administration of lactoferrin decreased the occurrence of colon cancer by 83%. The number of adenocarcinoma cells in the gut of rats was reduced after the ingestion of lactoferrin.

Lactoferrin-mediated inhibition of tumor growth might be related to apoptosis of these cells, induced by the activation of the Fas signaling pathway [55]. It has been suggested that the treatment of lactoferrin knockout mice with lactoferrin (post-chemotherapy) accelerated the reconstitution of the immune system. This also reduced the chances of infection following chemotherapy treatment [76]. Lactoferrin can scavenge free iron in fluids and inflamed and infected sites, suppressing free radicalmediated damage and decreasing the availability of the metal to pathogens and cancer cells. Also, lactoferrin hinders migration in a model of human glioblastoma by reverting an epithelial-to-mesenchymal transition-like process [4, 32].

#### **3.4 Lactoferrin in COVID-19 treatment**

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has recently become a primary global health concern, leading to the urgent development of therapeutic agents for its prevention and treatment. Iron overload is understood to have an important role in the pathogenesis of COVID-19. Actually some features (such as inflammation hyperferritinemia, hypercoagulation, and immune dysfunction) manifested a in COVID-19, are linked to iron overload. The presence of free iron, resulting from iron overload and dysregulation, is very highly reactive and toxic due to its reactive oxygen species (ROS) generation potential. The ROS produced react with very important cellular biomolecules and induce their subsequent damage. Nucleic acids, proteins as well as membranous and cellular lipids are effected by the highly activate inflammatory processes which may be either acute or chronic. The linkage of inflammation with multiple clinical conditions, such as cancer is well understood [80]. Lactoferrin has exhibited unique immunomodulatory, anti-inflammatory, and broadspectrum antiviral activity indicating its potential for the cure of COVID-19 cases and prevention of its devastating effects on multiple target organs [81, 82]. Lactoferrin could counteract the coronavirus infection and inflammation, acting either as a natural barrier of respiratory and intestinal mucosa or reverting the iron disorders related to the viral colonization. Iron-catalyzed lipid damage is understood to exerts a direct effect on ferroptosis, the newly discovered cell death mechanism. Unlike programmed cell death (PCD), ferroptosis not only leads to amplified cell death but is also associated with inflammation. Iron chelators are generally recognized as safe and have been shown to protect patients in diseases characterized by iron overload. Research work also suggests that iron chelators exhibit antimicrobial activities. It is suggested that the naturally occurring iron chelators, such as lactoferrin, exert anti-inflammatory

#### *Medicinal Potential of Camel Milk Lactoferrin DOI: http://dx.doi.org/10.5772/intechopen.108316*

as well as immunomodulatory effects. It binds to some of the same receptors used by coronaviruses and hence blocks its entry into host cells. Iron chelators may actually be of a very high therapeutic value during the present scenario of the ongoing COVID-19 pandemic [80]. Therefore, the use of lactoferrin may be of value in the prevention and management of COVID-19. The use of lactoferrin appears to be a promising approach to treating COVID-19, but further investigations are required to verify its antiviral activity *in vitro* and *in vivo* [83–85]*.*

#### **3.5 Lactoferrin assimilation** *in vivo*

Lactoferrin shows high bioavailability after oral administration, high selectivity toward cancer cells, and a wide range of molecular targets controlling tumor proliferation, survival, migration, invasion, and metastasis. Notably, lactoferrin may either promote or inhibit cell proliferation and migration depending on whether its target cell is normal or cancerous. Significantly, its administration is well tolerated and does not exhibit any significant side effects. Furthermore, lactoferrin may prevent cancer development and growth by enhancing the adaptive immune response. Oral administration of lactoferrin has also led to promising improvement in the immune responses of antiretroviral therapy in naıve children suffering from HIV [86]. Oral administration of lactoferrin decreased the occurrence of colon cancer by 83%, while the quantity of adenocarcinoma cells was reduced in the gut of rats after ingestion of Lactoferrin, ameliorating tongue cancer.

Of particular interest is the notion that even its oral administration may be effective. This is different from many other therapeutic proteins, which typically require other invasive routes of administration [87]. Oral administration of bovine lactoferrin prevents carcinogenesis in the colon and other organs in rats. It also inhibits lung metastasis in mice. It might be mediating its anti-carcinogenesis effects is by increasing expression of relevant cytokines and inducing subsequent activation of immune cells [67]. It interacts with a wide range of molecular targets controlling tumor proliferation, survival, migration, invasion, and metastasis. It may be noted that lactoferrin can promote or inhibit cell proliferation and migration depending on whether it acts upon normal or cancerous cells, respectively. Moreover, lactoferrin can prevent the development or inhibit cancer growth by boosting adaptive immune response. Most importantly, lactoferrin administration is highly tolerated and does not present significant adverse effects.

Oral administration of lactoferrin is the most widely adopted method of its delivery into the human body. This still possesses some challenges that must be addressed before reaping the highest benefit from its intake. Since the functional domains of lactoferrin are highly dependent on its unique 3D structural conformation, the gastrointestinal breakdown of lactoferrin may cause undesirable loss of some of its functional properties. The important receptors of lactoferrin are located at the intestinal mucosa and lymphatic tissue cells in the gut [88–91]. Hence, the delivery of lactoferrin through oral administration requires that it is protected so that it passes through the stomach and is delivered to the absorption sites in a functionally active form. But the most important thing is to note that the digestive tract in infants and newborns is not mature enough (e.g., the intragastric pH and the gastric emptying rate are higher than in adults), and lactoferrin would not be completely digested under these conditions. This hypothesis has been confirmed by measuring the unhydrolyzed lactoferrin in fecal extracts of babies [92, 93]. Nevertheless, the degradation of lactoferrin during the gastrointestinal tract could also be beneficial. It has been reported that

strong antibacterial peptides such as lactoferricin and lactoferrampin are produced by its pepsin hydrolysis [94, 95]. This further benefits the utilization of lactoferrin in high value food products such as infant formula, nutritional supplements, and other formulations that aim at delivering lactoferrin through oral administration.

A commonly accepted method to protect lactoferrin during digestion is microencapsulation. In this method, a protective matrix is created around the lactoferrin core. Food grade proteins (e.g., bovine serum albumin, β- lactoglobulin) and polysaccharides (e.g., pectin, carrageenan, sodium alginate, gum Arabic) are commonly used as the shell materials. This core-shell structure excellently protects lactoferrin from the harsh environment prevailing in the human digestive system. The microencapsulation also helps achieve targeted and controlled release of lactoferrin by simply using shell materials with suitable properties.

Based on in vivo studies, oral administration of lactoferrin to rodents significantly decreased the chemically induced carcinogenesis in various organs such as the breast, esophagus, tongue, lung, liver, colon, bladder, and hindered angiogenesis [78]. During the past two decades, many animal and human studies have proved that orally administered Lactoferrin exerts many beneficial effects on the health of animals and humans [75].

#### **3.6 Lactoferrin industries in the world**

Human and bovine lactoferrin is generally recognized as a safe substance (GRAS) by the Food and Drug Administration (FDA, USA). Some pharmaceutical industries (*e.g.*, Morinaga Milk Industry Co LTD Venture LLC, Ventria Bioscience, AusBioMed, Biopharming, Max Biocare, etc.) are into commercialization of human and bovine Lactoferrin related products such as nutraceuticals and vitamin supplements for pediatric use. Also are being produced baby foods, beverages, and a cell growth promoting adjuncts for better child development.

According to Global Market Insights Inc. report, the global lactoferrin powder revenue size was US\$195 million (€164 m) in 2020, which is set to surpass US\$315 million (€364.1 m) by 2027 and is expected to register over 7.7% CAGR between 2021 and 2027. Owing to the anti-inflammatory attribute of lactoferrin its market is likely to surpass 70 Million USD by 2027. Its antiviral efficacy is being increasingly recognized during the COVID-19 pandemic. Furthermore, its immunomodulatory and anti-inflammatory capability is expected to raise product demand in an unprecedented manner from the pharmaceutical sector. It is estimated that the lactoferrin industry from the pharmaceutical application would actually exceed 53.78 Million USD by 2027. The global Lactoferrin Market is anticipated to attain substantial growth by the end of the forecast period (2021–2025).

Lactoferrin has also been used in different products, such as probiotics, supplemental tablets, cosmetics, and as a natural solubilizer of iron in food. It is also used in the treatment of diverse carcinomas, severe sepsis, and diabetic foot ulcers. Numerous attributes of lactoferrin, such as its iron absorption ability, antibacterial, anti-inflammatory, antioxidant, and immunity-boosting capabilities, are likely to provide promising opportunities for the lactoferrin industry. The ability of lactoferrin to prevent biofilm formation helps inhibiting the growth of bacteria. This can lead to an enhanced product demand owing to its therapeutic applications. The higher susceptibility of infections in infants and newborns due to an underdeveloped immune system can be supplemented by the lactoferrin industry. Growing demand for lactoferrin from physical fitness and sports nutrition application is likely to drive the growth of

lactoferrin capsules during the forecast period. Increasing instances of digestive and gastric disorders should boost the demand for lactoferrin as an anti-inflammatory ingredient.
