**3.4 Other promising biological activities**

Biosurfactants have been shown to have many other roles in biomedical application. Some of the most powerful molecules (eg. surfactin, mannosylerythritol lipids (MELs), trehalose lipids) are known to have anti-inflammatory, anti-tumour, immunosuppressive and immunomodulating functions, in addition to other properties such as self-assembling, human cells stimulation and differentiation, interaction with stratum corneum lipids, cell-tocell signaling, hemolytic activity.

## **3.4.1 Anti-tumor activity**

Recently, it has been demonstrated that these interesting microbial products can control a variety of mammalian cell functions. They are considered to participate in various intercellular molecular recognitions such as signal transduction, cell differentiation, cell immune response, etc. (Osada, 1998). Cao et al., (2010) demonstrated that surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. In a more recent work, they investigated the reactive oxygen species (ROS) and Ca2+ impact on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis induced by surfactin (Cao et al., 2011). The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential which lead to an increase in the cytoplasmic Ca2+ concentration. In addition, cytochrome c was released from mitochondria to cytoplasm through the MPTP which activated caspase-9, eventually inducing apoptosis.

In another study, viscosin, an effective surface-active cyclic lipopeptide recovered from *Pseudomonas libanensis* M9-3, inhibited the migration of the metastatic prostate cancer cell line, PC-3M, without visible toxicity effects (Saini et al., 2008). More recently, lipopeptides (namely isoforms of surfactins and fengycins) derived from a marine *Bacillus circulans* DMS-

suggesting that it could be safely used as a therapeutic agent or as a preservative in food or

In another recent work, a biosurfactant producing strain, *Lactobacillus* sp. CV8LAC, isolated from fresh cabbage, showed interesting antiadhesive activity against two *C. albicans* pathogenic biofilm-producing strains (CA-2894 and DSMZ 11225) (Fracchia et al., 2010). The CV8LAC biosurfactant significantly inhibited the adhesion of fungal pathogens to polystyrene microtiter plates in pre-coating and co-incubation experiments. In pre-coating assays, biofilm formation of strain CA-2894 was reduced by 82% at concentration of 312.5 µg/mL while that of strain DSMZ 11225 was reduced by 81% at 625 µg/mL. In co-incubation assays, biofilm formation of the two strains was inhibited by 70% at 160.5 µg/well and by 81% at 19.95 µg/well, respectively. It was interesting to note that no inhibition of both *C. albicans* planktonic cells was observed, thus indicating that the

biosurfactant displayed specific anti-biofilm formation but not antimicrobial activity.

the pathogenesis of nosocomial infections (Falagas & Makris, 2009).

**3.4 Other promising biological activities** 

cell signaling, hemolytic activity.

**3.4.1 Anti-tumor activity** 

inducing apoptosis.

Considering their importance for human health and their recognized safety, environmental probiotic organisms may, thus, represent a safe and effective intervention for infection control purposes. Probiotics themselves or their products (biosurfactants), could be applied to patient care equipment, such as tubes or catheters, with the aim of decreasing the colonisation of these sites by nosocomial pathogens and potentially impede a central step in

Biosurfactants have been shown to have many other roles in biomedical application. Some of the most powerful molecules (eg. surfactin, mannosylerythritol lipids (MELs), trehalose lipids) are known to have anti-inflammatory, anti-tumour, immunosuppressive and immunomodulating functions, in addition to other properties such as self-assembling, human cells stimulation and differentiation, interaction with stratum corneum lipids, cell-to-

Recently, it has been demonstrated that these interesting microbial products can control a variety of mammalian cell functions. They are considered to participate in various intercellular molecular recognitions such as signal transduction, cell differentiation, cell immune response, etc. (Osada, 1998). Cao et al., (2010) demonstrated that surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. In a more recent work, they investigated the reactive oxygen species (ROS) and Ca2+ impact on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis induced by surfactin (Cao et al., 2011). The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential which lead to an increase in the cytoplasmic Ca2+ concentration. In addition, cytochrome c was released from mitochondria to cytoplasm through the MPTP which activated caspase-9, eventually

In another study, viscosin, an effective surface-active cyclic lipopeptide recovered from *Pseudomonas libanensis* M9-3, inhibited the migration of the metastatic prostate cancer cell line, PC-3M, without visible toxicity effects (Saini et al., 2008). More recently, lipopeptides (namely isoforms of surfactins and fengycins) derived from a marine *Bacillus circulans* DMS-

cosmetic products.

2 showed interesting cytotoxic activity against cancer cell lines (Sivapathasekaran et al., 2010). The purified lipopeptides at a concentration of 300 µg/mL showed more than 90% inhibition of proliferation on both colon cancer cell lines HCT 15 and HT 29 after 24 h treatment and the antiproliferative activity of lipopeptides was observed in a dose dependent manner.

Significant effects against both tumor cell lines as compared to non-tumor cell line were also observed, thus indicating the selective inhibitory activity of these molecules. Serratamolide AT514, cyclic depsipeptide from *Serratia marcescens,* belonging to the group of serrawettins, has also been reported to be a potent inducer of apoptosis of several cell lines derived from various human tumors and B-chronic lymphocytic leukemia cells, primarily involving the mitochondria-mediated apoptotic pathway and interference with Akt/NF-kB survival signals (Escobar-Díaz et al., 2005, as cited in Matsuyama et al., 2010). Biological studies of AT514 using human B-lymphocytes are now in progress for clinical applications of AT514 in the field of medical oncology.

Interesting anti-tumor activities has also been reported for glycolipids. Mannosylerythritol lipids (MELs) are among the most promising biosurfactants known due to their versatile interfacial and biochemical actions. Interesting studies, thoroughly reviewed by Kitamoto et al. (2002) and by Arutchelvi & Doble, (2010), have shown that MEL-A and MEL-B display excellent growth inhibition and differentiation-inducing activities against human leukemia cells including myelogenous leukemia cell K562, promyelocytic leukemia cell HL60, and the human basophilic leukemia cell line KU812, as well as growth inhibition activity of mouse melanoma B 16 cells. Recently Chen et al., (2006) also demonstrated that a sophorolipid produced from the yeast *Wickerhamiella domercqiae* induced apoptosis in H7402 human liver cancer cells by blocking cell cycle at G1 phase and partly at S phase, activating caspase-3, and increasing Ca2+ concentration in cytoplasm.

### **3.4.2 Anti-inflamatory activity**

Byeon et al., (2008) observed that surfactin was able to downregulate LPS-induced nitric oxide production in RAW264.7 cells and primary macrophages by inhibiting NF-κB activation, suggesting a good potential as a bacterium-derived anti-inflammatory agent. Selvam et al., (2009) studied the effect of *B. subtilis* PB6, a natural probiotic, on plasma cytokine levels in inflammatory bowel disease and colon mucosal inflammation. The strain was found to secrete surfactins which are known to inhibit phospholipase A2, involved in the pathophysiology of inflammatory bowel disease. In animal experiments carried out in rat models for trinitrobenzene sulfonic acid-induced colitis, oral administration of PB6 as a probiotic suppressed colitis as measured by mortality rate and changes in colon morphology and weight gain. Plasma levels of pro-inflammatory cytokines were also significantly lowered and the anti-inflammatory cytokine significantly increased after the oral administration of PB6, supporting the concept that PB6 inhibits PLA2 by secreting surfactins.

In another work, surfactin isomers derived from the mangrove bacterium *Bacillus* sp. (No. 061341) showed interesting anti-inflammatory activities (Tang et al., 2010). In particular, this class of cyclic lipopeptide showed strong inhibitory properties on the overproduction of nitric oxide and the release of IL-6 in LPS-induced murine macrophage cell RAW264.7. Moreover, structure-activity relationship studies revealed that the existence of the free carboxyl group in the structure of surfactin isomer was crucial as to the anti-inflammatory activities. An interesting recent study explored the mechanisms responsible for surfactininduced anti-inflammatory actions in the context of periodontitis caused by *Porphyromonas*  *gingivalis,* the major pathogen of periodontal disease (Park et al., 2010). The Authors observed that surfactin significantly reduces pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-12, through suppression of nuclear factorκB activity in *P. gingivalis* LPS-stimulated THP-1 human macrophage cells, in a Heme oxygenase-1 (HO-1)-dependent fashion. Furthermore, surfactin treatment effectively induces HO-1 expression, a major defense in response to oxidative stress.

These observations support the potential of surfactin as a candidate in strategies to prevent caries, periodontitis, or other inflammatory diseases.

## **3.4.3 Immuno-modulatory action**

Park & Kim, (2009) studied the role of surfactin in the inhibition of the immunostimulatory function of macrophages through blocking the NK-κB, MAPK and Akt pathway. This provided a new insight into the immunopharmacological role of surfactin in autoimmune disease and transplantation. Their work indicated that surfactin has potent immunosuppressive capabilities which suggested important therapeutic implications for transplantation and autoimmune diseases, including allergy, arthritis and diabetes.

A biosurfactant glycolipid complex from *Rhodococcus ruber* was also shown to activate the production of IL-1beta and TNF-alpha cytokines without modifying the production of IL-6, thus suggesting good prospects for further studies of immunomodulating and antitumor activities (Kuyukina et al., 2007).

### **3.4.4 Other biomedical related properties**

Han et al., (2008) observed that high surfactin micelle concentration affected the aggregation of amyloid β-peptide (Aβ (1-40)) into fibrils, a key pathological process associated with Alzheimer's disease. Fengycin, another lipopeptide biosurfactant is also able to cause membrane perturbations (Deleu et al., 2008). Recent results by Eeman et al., (2009) emphasized the ability of fengycin to interact with the lipid constituents of the stratum corneum extracellular matrix and with cholesterol. Another interesting property of surfactin and its synthetic analogues is the ability to alter the nanoscale organisation of supported bilayers and to induce nanoripple structures with intriguing perspectives for biomedical and biotechnological applications (Bouffioux et al., 2007; Brasseur et al., 2007; Francius et al., 2008). Morita et al., (2010) investigated the cell activating property of MELs using cultured fibroblast and papilla cells, and a three dimensional cultured human skin model. The diacetylated MEL (MEL-A) produced from soybean oil significantly increased the viability of the fibroblast and of the papilla cells over 150% compared with that of control cells, suggesting potential use as new hair growth agent stimulating the papilla cells. Using a three-dimensional cultured human skin model, Morita et al., (2009b) observed that the viability of the SDS damaged cells was markedly improved by the addition of MEL-A in a dose-dependent manner. This demonstrated that MEL-A also had a ceramide-like moisturising activity toward the skin cells. Similarly, (Kitagawa et al., 2007, as cited in Worakitkanchanakul et al., 2008) reported that MEL-B shows excellent moisturizing properties, equivalent to those of natural ceramides, toward human skin.

Trehalose lipids also display various interesting biological activities mainly due to their great tendency to partition into phospholipid membranes (Ortiz et al., 2008, 2009). In particular, the trehalose lipid was suggested to incorporate into the membrane bilayers and produce structural perturbations, which might affect the function of both phosphatidylethanolamine and phosphatidylserine membranes. Zaragoza et al., (2010) observed that a succinoyl trehalose

*gingivalis,* the major pathogen of periodontal disease (Park et al., 2010). The Authors observed that surfactin significantly reduces pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-12, through suppression of nuclear factorκB activity in *P. gingivalis* LPS-stimulated THP-1 human macrophage cells, in a Heme oxygenase-1 (HO-1)-dependent fashion. Furthermore, surfactin treatment effectively

These observations support the potential of surfactin as a candidate in strategies to prevent

Park & Kim, (2009) studied the role of surfactin in the inhibition of the immunostimulatory function of macrophages through blocking the NK-κB, MAPK and Akt pathway. This provided a new insight into the immunopharmacological role of surfactin in autoimmune disease and transplantation. Their work indicated that surfactin has potent immunosuppressive capabilities which suggested important therapeutic implications for

A biosurfactant glycolipid complex from *Rhodococcus ruber* was also shown to activate the production of IL-1beta and TNF-alpha cytokines without modifying the production of IL-6, thus suggesting good prospects for further studies of immunomodulating and antitumor

Han et al., (2008) observed that high surfactin micelle concentration affected the aggregation of amyloid β-peptide (Aβ (1-40)) into fibrils, a key pathological process associated with Alzheimer's disease. Fengycin, another lipopeptide biosurfactant is also able to cause membrane perturbations (Deleu et al., 2008). Recent results by Eeman et al., (2009) emphasized the ability of fengycin to interact with the lipid constituents of the stratum corneum extracellular matrix and with cholesterol. Another interesting property of surfactin and its synthetic analogues is the ability to alter the nanoscale organisation of supported bilayers and to induce nanoripple structures with intriguing perspectives for biomedical and biotechnological applications (Bouffioux et al., 2007; Brasseur et al., 2007; Francius et al., 2008). Morita et al., (2010) investigated the cell activating property of MELs using cultured fibroblast and papilla cells, and a three dimensional cultured human skin model. The diacetylated MEL (MEL-A) produced from soybean oil significantly increased the viability of the fibroblast and of the papilla cells over 150% compared with that of control cells, suggesting potential use as new hair growth agent stimulating the papilla cells. Using a three-dimensional cultured human skin model, Morita et al., (2009b) observed that the viability of the SDS damaged cells was markedly improved by the addition of MEL-A in a dose-dependent manner. This demonstrated that MEL-A also had a ceramide-like moisturising activity toward the skin cells. Similarly, (Kitagawa et al., 2007, as cited in Worakitkanchanakul et al., 2008) reported that MEL-B shows excellent moisturizing

transplantation and autoimmune diseases, including allergy, arthritis and diabetes.

properties, equivalent to those of natural ceramides, toward human skin.

Trehalose lipids also display various interesting biological activities mainly due to their great tendency to partition into phospholipid membranes (Ortiz et al., 2008, 2009). In particular, the trehalose lipid was suggested to incorporate into the membrane bilayers and produce structural perturbations, which might affect the function of both phosphatidylethanolamine and phosphatidylserine membranes. Zaragoza et al., (2010) observed that a succinoyl trehalose

induces HO-1 expression, a major defense in response to oxidative stress.

caries, periodontitis, or other inflammatory diseases.

**3.4.3 Immuno-modulatory action** 

activities (Kuyukina et al., 2007).

**3.4.4 Other biomedical related properties** 

lipid produced by *Rhodococcus* sp. caused the swelling of human erythrocytes followed by hemolysis at concentrations well below its critical micellar concentration. They concluded that trehalose lipid caused the hemolysis of human erythrocytes by a colloid-osmotic mechanism, most likely by formation of enhanced permeability domains, or "pores" enriched with biosurfactant, within the erythrocyte membrane.

Permealization of biological and artificial membranes was also reported to be induced by *Pseudomonas aeruginosa* dirhamnolipid (Sánchez et al., 2010). In particular, it caused the hemolysis of human erythrocytes through a lytic mechanism, as shown by the similar rates of K+ and hemoglobin leakage, and by the absence of effect of osmotic protectants. Scanning electron microscopy showed that the addition of the biosurfactant changed the usual disc shape of erythrocytes into that of spheroechinocytes.
