Candida bombicola NRRL Y-17069 was obtained from the Agricultural Research Service, Peoria, Illinois, USA. The culture was kept in a stock slant medium (Wickerman, 1951).

Sunflower seeds (Giza 1) were obtained from the local market (Cairo, Egypt). The seeds were pressed with laboratory-type Carver hydraulic press under 10.000 Ib in⁻² pressure for 1 h at room temperature according to Üstun et al. (1990), then the sunflower oil cake residue was collected, frozen and kept at -4 °C until analysis. Crude soybean oil was obtained from the Food Technology Research Institute, Soy Processing Center, Agriculture Research Center, Giza, Egypt. All chemicals and reagents used were of analytical grade.

The inoculum was prepared by transferring a loopful of a stock culture (7 days old) of C. bombicola to a 50 ml sterile inoculum medium (Wickerman, 1951), which was then incubated at 30 °C, 180 rpm for 24h.

Surface tension and critical micelle dilution (CMD) were determined with Du-Nouy tensiometer (Kruss type 8451). CMD is defined as the reciprocal of the SL dilution at which a sharp increase in surface tension is observed. To determine the CMD, different serial dilutions were done for the SL extracts (EI, EII, and EIII) obtained from C. bombicola (McInerney et al., 2004).

The infrared (IR) spectrum (from 400 to 4000 wave numbers, cm⁻¹) of SL extracts were recorded using a KBr pellet in Nicolet Impact 6100 FTIR spectrophotometer JASCO, USA.

The NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer. ¹H spectra were run at 300 MHz in deuterated chloroform (CDCl₃). Chemical shifts are quoted in δ and were related to those of the solvents.

Anticancer activity screening for the tested compounds with 4 different human tumor cell lines including breast adenocarcinoma MCF-7; hepatocellular carcinoma HepG2, lung cancer A549 and colon cancer HCT116 which were obtained from the American Type Culture Collection (Rockville, MD, USA). The tumor cells were kept in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (GIBCO), peni cillin (100 U·mL⁻¹) and streptomycin (100 µg·mL⁻¹) at 37 °C in a humidified atmosphere containing 5% CO₂. Cells at a concentration of 0.50×10⁶ were grown in a 25 cm² flask in 5 mL of a complete culture medium.

The anti-proliferative activity was measured in vitro using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay according to the previously reported standard procedure (Mosmann 1983). The absorbance was measured with a microplate reader (AsysHitech, Austria) at 570 nm. The relationship between surviving fraction and compound concentration was plotted to get the survival curve for each cell line after the specified time. The concentration required for 50% inhibition of cell viability (IC₅₀) was calculated.

The level of urokinase (uPA) protein expression was determined using AssayMax human uPA ELISA kit (Assaypro, USA) according to manufacturer's instructions. The prepared compounds as well as a standard drug, doxorubicin were incubated for 48 h with, HepG2 and A549 cells at a concentration of 1/10 of the IC₅₀ values of each compound.

Histone deacetylase inhibitors represent a promising new class of compounds for the treatment of cancer (Walkinshaw and Yang, 2008). The activity of HDAC in the lysate of hepatic HepG2 and lung A549 cancer cells treated with the prepared compounds was measured using a colorimetric assay kit (BioVision, Mountain View, kit no. K331-100).

The results are reported as Mean±Standard error (S.E.) for experiments repeated at least four times. Statistical differences were analyzed according to the one way ANOVA test followed by the student's t test wherein the differences were considered to be significant at p<0.05.

Utilization of a local waste for a maximum production of crude biosurfactant with low cost production materials and processing was the main purpose of this work. Different fermentation techniques were used in this study. By using a submerged medium (medium I), the crude SL obtained reached the level of 20.6 g·100 g⁻¹ substrate by extraction with ethyl acetate from the substrates (sunflower oil cake plus soybean oil) on the fourth day of fermentation (Figure 1). While a non-significant amount of SL was produced from the same medium without soybean oil (medium II). The yield obtained from the medium I (41.2 g·L⁻¹) was higher than that of Davery and Pakshirajan (2010), who found that the maximum yield of C. bombicola SL ranged from 23.3 to 33.3 g·L⁻¹ using mixed hydrophilic substrates. Also, it was higher than that obtained by Parekh et al. (2012) (5.8 g·100 g⁻¹ substrate) using C. Starmerella grown in submerged media containing various lipids and fatty acids obtained from mango kernel. On the other hand, the present yield was slightly lower than that produced by Li et al. 2013 (47 g·L⁻¹) and Sudha et al., 2010 (49.8 g·L⁻¹) using Wickerhamiella domercqiae var. and C. tropicalis respectively. By using SSF for the same substrates, which consists of sunflower oil cake plus soybean oil (medium III), different extraction methods were examined in one step extraction. Using methanol as extraction solvent, the extract (E I) gave a yield of SL 17 g·100 g⁻¹ substrate, while the extraction by ethyl acetate gave a yield of 22 g·100 g⁻¹ substrate. However, using a mixture of methanol and ethyl acetate at a ratio of 1:1 (in one step extraction), the yield was 15 g·100 g⁻¹ substrate.

On the other hand, employing a new concept for the production process by re-extraction of the remaining fermented media (after isolation of EI) with ethyl acetate (E II) resulted in a higher additional amount of the produced SL (32.5 g·100 g⁻¹) as shown in Figure 1. This new method of re-extraction produced a higher yield of SL than the traditional extraction method for the solid medium using methanol alone, whereas the two extracts (E I and E II) produced a level of 49.5 g·100 g⁻¹ for the same substrates on the fourth day of fermentation.

Parekh et al. (2012) found that the SL obtained from C. Starmerella with SSF of mango kernel was 17.48 g·100 g⁻¹ substrate after extraction by ethyl acetate, which was lower than our results for ethyl acetate extraction alone (22 g·100 g⁻¹ substrate). On the other hand, the total yields obtained in this study from methanol extract EI (17 g·100 g⁻¹ substrate) and ethyl acetate re-extract EII (32.5 g·100 g⁻¹ substrate) were significantly higher (49.5 g·100 g⁻¹ substrate).

The highest yield obtained from medium III using our modified re-extraction method was confirmed to be the most suitable extraction method used. From all the above results, it can be concluded that the SSF technique is a suitable technique for further studies with the two steps of extraction.

The course of time of SL production during the SSF (medium III) using the modified extraction method investigated in this study is presented in Figure 2. It can be observed that the methanol extract (E I) productivity was directly proportional with incubation time, whereas the higher yield of SL was obtained on the 16^th and 12^th days, giving 28.75 and 28.11 g·100 g⁻¹ substrate, respectively; while, lower yields were achieved on the 4^th and 8^th days of incubation (17 and 20.52 g·100 g⁻¹ substrate, respectively). However, the ethyl acetate re-extract (E II) productivity was inversely proportional with incubation time, where the higher SL yield was obtained on the 4^th and 8^th days (32.5 and 22.13 g·100 g⁻¹ substrate, respectively); subsequently the lower yield was produced on the16^th and 12^th days of fermentation (10.5 and 12.8 g·100 g⁻¹ substrate, respectively). The highest yield obtained from both extracts over the course of fermentation time was 49.5 g·100 g⁻¹ substrate on the 4^th day, while the lowest was 39.25 g·100 g⁻¹ substrate on the 16^th day of incubation.

One of the important characterization properties of a potent surfactant is its ability to lower the surface tension in aqueous solutions. CMD is an indirect indication of surfactant concentration (Makkar and Cameotra, 1997). So, the surface tension (S.T.) and critical micelle dilution (CMD) were determined to evaluate the surface activity of the produced SL resulting from both solvents (E I and E II) during the fermentation period. It was found that the SL extracted with methanol (E I) on the 12^th day showed the highest efficiency in reducing the surface tension of water from 70 to 45 mN·m⁻¹ when the sample was diluted to 0.1X (10% concentration) which corresponds to its CMD, followed by the ethyl acetate extract (E II) on the same day which gave 48 mN·m⁻¹ at the same dilution (0.1X) of 10% concentration CMD. This ethyl acetate extract (E II) was partially purified by hexane to produce the E III extract, which was found to have a non-significant effect on reducing the surface tension (47 mN·m⁻¹) compared to the E II extract at the same CMD (Figure 3). However, the surface tension of the other SL extracts using both solvents during the fermentation period (4–16 days) ranged from 51–56 mN·m⁻¹.

In light of the above results, more studies will be done on the compounds (E I, E II and E III) on the 12^th day of fermentation. These results of S.T. for E I & E II nearly coincide with those reported by Kim et al. (2005), in that the S.T. of C. bombic ola SL was 48 mN·m⁻¹ when grown in a medium containing glucose and dark soybean oil as carbon sources. However, they were lower than those stated by Amaral et al. (2006), who found that the biosurfactant from Yarrowia lipolytica has the ability to reduce S.T. by up to 50 mN·m⁻¹

On the other hand, the prepared SL exhibited a higher minimum S.T. than that produced from many candida species and ranged from 32–39 mN·m⁻¹ (Sobrinho et al., 2008; Daverey and Pakshirajan 2009, 2010; Chandran and Das 2011).

The FT-IR spectra of the prepared SL compounds (E I, E II and E III) showed mostly identical absorption bands (Figure 4). The FT-IR spectra for all extracts revealed broad bands at 3385–3424 cm⁻¹ which correspond to the O-H stretch, while asymmetrical stretching (v_as CH₂) and symmetrical stretching (v_s CH₂) of methylene occurs at 2926 cm⁻¹ and 2855 cm⁻¹ respectively. Absorption bands at (1742–1745 cm⁻¹) contribute to C=O stretching from lactone ester or acids. The C=O absorption band from acetyl esters was observed at 1238–1240 cm⁻¹ in all structures, while the bands at 1460 cm⁻¹ corresponded to the C-O-H in the plane binding of carboxylic acid (-COOH) in the structure of the three products. The stretch of C-O band of C (-O)-O-C in lactones exists in all products at 1164–1170 cm⁻¹, also the C-O stretch from C-O-H groups of sugar (sophorose moiety) is observed at 1080–1098 cm⁻¹; finally the IR spectra revealed the absorption band for C=C at 719–723 cm⁻¹ in all the studied structures. All these structural details of the products were found similar to those reported by Yongmei and Lu-Kwang (2001); Daverey and Pakshiragan (2009) and Bajaj et al. (2012), which confirmed that these products are SL compounds.

The structure of the produced SL compounds (E I, E II and E III) was elucidated using ¹H NMR analysis. The spectra of the three extracts were assigned to a typical glycolipid-type structure. In all compounds, the signals of the vinyl group (–CH=CH–) were at 5.32≈5.39 ppm, while the signals at 2.08 ppm were denoting the presence of–CH₃ group. Multiple peaks from 1.23 to 1.31 ppm put the accent on the existence of fatty acid chain moiety in all structures. The presence of the–CO-CH₃ group in all structures was confirmed by the signals at 2.06 ppm in the spectra; on the other hand, the protons of two glucose were resonated at 3.4≈4.3 ppm in the Hexane fraction (E III). The signals obtained were similar to the one in the ¹H NMR spectrum of the SL compounds reported by other researchers (Wei et al., 2005; Chen et al., 2006a; Gao et al., 2007; Daverey and Pakshiragan 2009 and Bajaj et al., 2012).

According to the results of the FT-IR and ¹H NMR analyses, all these structural features further confirmed the SL group of compounds (acidic and lactone ring form) in the produced biosurfactants.

The anti-proliferative activity of the produced crude and partially purified SLs (E l, E II and E III) and a mixture of E I and E III (E IV) was evaluated against breast adenocarcinoma MCF-7; human hepatocellular carcinoma HepG2, human lung cancer A549 and human colon cancer HCT116 cell lines using an MTT colorimetric assay, in comparison with doxorubicin as reference drug. The results revealed that all compounds did not exert any activity against human breast cancer MCF-7 or colon cancer HCT116 cell lines.

The anti-proliferative activities are expressed by median growth inhibitory concentration (IC₅₀) and provided in Table 1. From the results it is evident that the tested SLs compounds displayed potent growth inhibitory activity against only two tested cell lines (HepG2 and A549).

The IC₅₀ of compounds E l, E II, E III and E IV for the HepG2 cell line was 47.00±4.20, 36.00±3.50, 23.70±2.63 and 19.00±1.80 µg·mL⁻¹ respectively. It is clear that, while the IC₅₀ of E III (23.70±2.63 µg·mL⁻¹) is close to the value of the reference drug, doxorubicin (20.10±2.00 µg·mL⁻¹), the mixture of the 2 extracts (E IV) was more potent (19.00±1.80 µg·mL⁻¹) than the doxorubicin. Similarly, the IC₅₀ of SL compounds E I, E II, E III and E IV for the A549 cell line was 46.60±4.30, 41.20±4.60, 29.00±2.80 and 25.77±2.20 µg·mL⁻¹ respectively. It is clear that the mixture (E IV) was more potent (25.77±2.20 µg·mL⁻¹) than doxorubicin (25.50±2.70 µg·mL⁻¹).

To identify the mechanism of action responsible for the cytotoxicity of the prepared SL compounds, the level of urokinase (uPA) protein expressed in the two cell lines (HepG2 and A549) were estimated quantitatively.

In the case of the hepatic cancer cell line HepG2 and lung cancer cell line A549, the SL compounds E I and E II have no effect on the expression of uPA, only 4 and 3% inhibition were obtained in HepG2, while 2% and 2% inhibition were obtained in A549 cells. On the other hand, the level of uPA expression decreased in SL compounds E III and E IV by the following percent, E III (65, 55%) and E IV (75, 63%) in HepG2 and A549, respectively. From the results, the sophorolipid compound E IV exhibited a good anticancer activity in HepG2 and A549 similar to doxorubicin (92% and 86%, respectively) (Figure 5). In both HepG2 and A549 the inhibition of uPA activity of the tested SLs compounds was in accordance with the cytotoxicity activity.

Histone deacetylase (HDAC) inhibitors are a new class of targeted anticancer agents which are potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo (Bi and Jiang, 2006). Thereby, we examined the effect of the prepared SL compounds as histone deacetylase inhibitors in an attempt to use them in tumor treatment.

In this work the activity of HDAC in the lysate of hepatic HepG2 and lung A549 cancer cells treated with the prepared SL compounds as well as Trichostatin, as a known inhibitor, was measured and the data were calculated as percentage of inhibition as compared to the control, untreated cancer cells. While the treatment of hepatic HepG2 and lung A549 cancer cells with Trichostatin resulted in 60% and 48% inhibition, respectively, as compared with the control untreated cancer cells, the treatment with compounds E l, E II, E III and E IV resulted in 6, 8, 38 and 47%, respectively in hepatic HepG2 cells. Similarly, the treatment of lung A549 cells with all the prepared SL compounds (E I-E IV) resulted in the inhibition of the activity of HDAC by 3, 5, 32 and 39%, respectively (Figure 6).

Taken together, these findings suggest that there are correlations between the cytotoxicity of the tested SL compounds and inhibition of the urokinase and Histone deacetylase activities. The tested compounds exert anti-carcinogenic activity in hepatic HepG2 and lung A549 cancer cell lines by lowering the activity of these enzymes which may reduce the cell proliferation and result in significant growth inhibition. The potent anticancer effect of the compound E IV needs further investigation.

Chen et al. (2006b) studied the cytotoxic effects of the SL produced by the yeast Wickerhamiella domercqiae on cancer cells of liver, lung and leukemia (H7402, A549, HL60 andK562). The results showed a dose-dependent inhibition ratio on cell viability according to the drug concentration ≤62.5 µg·mL⁻¹, which suggested that the SL produced by W. domercqiae have anticancer activity.

Fu et al. (2008) have demonstrated that acidic SL had stronger cytotoxicity (35.5% cytotoxicity at 100 mg mL concentration) than lactonic SL (26.2% cytotoxicity at 100 mg·mL⁻¹ concentration) against pancreatic cancer cells and he reported that these different results are probably attributed to the inhibition mechanism of SL on pancreatic cancer cells, which are different from other cancer cells. Also, Shao et al. (2012) investigated the bioactivity of SLs with different structures against human esophageal cancer cells, and the results revealed that the SLs with different unsaturation degrees of hydroxyl fatty acids had different cytotoxic effects on esophageal cancer cells.