Antioxidant activities and characterisation of polysaccharides isolated from the seeds of Lupinus angustifolius

Industrial Crops and Products 74 (2015) 950–956

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Antioxidant activities and characterisation of polysaccharides isolated from the seeds of Lupinus angustifolius Solomon Rajesh Thambiraj a , Michael Phillips a , Sundar Rao Koyyalamudi b,c , Narsimha Reddy a,∗ a

School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia Institute of Endocrinology and Diabetes, University of Sydney Medical School, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia c Discipline of Paediatrics and Child Health, University of Sydney Medical School, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia b

a r t i c l e

i n f o

Article history: Received 1 December 2014 Received in revised form 4 May 2015 Accepted 7 June 2015 Keywords: Lupinus angustifolius Polysaccharides Nutraceuticals Antioxidant activities Immunostimulatory effects

a b s t r a c t Lupinus angustifolius (blue lupin) is produced abundantly in Australia and used mainly as animal feed. However, these seeds have excellent nutritional value with high protein, high dietary fibre and low fat content with a huge potential for value addition. In the present study, blue lupin polysaccharides have been isolated and their biological activities investigated. Hot water extraction and size-exclusion chromatography yielded six polysaccharide fractions that have been designated as BLP-1, BLP-2, BLP-3, BLP-4, BLP-5 and BLP-6. Gas chromatography was employed to determine the mono-sugar compositions of these fractions which showed the existence of galactose, fucose, rhamnose, glucose, mannose, ribose and xylose. The antioxidant activities of all the fractions were investigated using ABTS+ * radical scavenging activity and ferrous chelating activity. Immunostimulatory activities of these fractions were measured by treating the mouse macrophages (RAW 264.7) and monitoring the production of nitric oxide (NO) by Griess reagent method. Three of the blue lupin polysaccharides (BLP-1, BLP-2 and BLP-5) displayed significant antioxidant activities. Some of the fractions have effectively stimulated mouse macrophages in a concentration-dependent manner indicating their immunostimulatory capability. FT-IR spectroscopic technique was employed for a preliminary structural characterisation of the active polysaccharide fractions. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Australia is world’s largest producer and exporter of Lupinus angustifolius (blue lupin) (Hofmanová et al., 2014). Lupin has been very valuable crop for Australian farmers in terms of rotational crop for nitrogen fixation in wheat fields and hence has become one of the major grain crops in the country. As a result, over 1 million tons of blue lupin grains are produced annually in Western Australia alone (Belski, 2012). Lupin crop also has global importance with significant production in Mediterranean area, South America and Europe (Martínez-Villaluenga et al., 2006). However, most of the lupin seeds are used for animal feed and less than 4% is consumed globally as human food (Belski, 2012). Considering the fact that lupin seeds have excellent nutritional value with high protein, high fibre (non-starch polysaccharides) and low fat content, there is a huge industrial potential for this produce (Sujak et al., 2006). Research on nutraceutical benefits of lupin

∗ Corresponding author. E-mail address: [email protected] (N. Reddy). http://dx.doi.org/10.1016/j.indcrop.2015.06.028 0926-6690/© 2015 Elsevier B.V. All rights reserved.

constituents will therefore be very valuable to food /nutraceutical industry and hence is expected to increase the market value for farmers. Lupin belongs to the family of Leguminosae and a large number of wild varieties of this plant exist (more than 200 species) (Erbas¸ et al., 2005). However, only four major species are cultivated worldwide, namely, L. angustifolius (blue lupin), Lupinus albus (white lupin), Lupinus luteus (yellow lupin) and Lupinus mutabulis (pearl lupin). These species are called sweet lupins owing to their low alkaloid content and hence are less toxic (Martínez-Villaluenga et al., 2006). The dominant variety of lupin produced in Australia is the L. angustifolius. Major health benefits of the sweet lupin based food include: lowering of blood pressure, improved bowel function, stimulation of growth of colonic microbiota, lower risk of colon cancer, controls blood glucose levels and improves cardiovascular health (Belski, 2012; Johnson et al., 2006; Lee et al., 2009; Smith et al., 2006; Yang et al., 2010). Lupin crop being the option for sustainable utilisation of land offers a great value to farmers. However, the tremendous health benefits of the grain produced from this crop are not fully utilised

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Fig. 1. Size-exclusion chromatogram of polysaccharide fractions from Lupinus angustifolius (blue lupin) (Fractions were collected in 95 tubes and the time increment between the tubes was 11 min. This corresponded to a total fractionation time of 17.42 h).

and further research to improve its industrial value is therefore necessary. It is clear from the literature (Lee et al., 2009; Duranti et al., 2008) that lupin is enriched with large contents of beneficial polysaccharides and protein. It is therefore possible to isolate these two important constituents and study their biological activities. A program of research has been initiated in authors’ laboratory towards this goal. It is well established that the polysaccharides derived from various botanical sources display high antioxidant, immunomodulatory, anti-cancer and several other important biological properties (Chen et al., 2014; Zheng et al., 2014; Zhong et al., 2012). Additionally, plant polysaccharides are known to exhibit least side effects (Zong et al., 2012). However, very limited literature is available on characterisation of polysaccharides isolated from lupins (Thumad Al-Kaisey and Wilkie, 1992). To the best of our knowledge there is no literature on biological activities of polysaccharides isolated from blue lupin. Considering their health benefits and abundance, it is extremely important to investigate the spectrum of biological activities that lupin polysaccharides display. This paper focusses on antioxidant and immunostimulatory activities of blue lupin polysaccharides and their structural characterisation. 2. Experimental 2.1. Materials and chemicals L. angustifolius seeds were obtained from Coorow seeds, Coorow, Western Australia, Australia. Sepharose CL-6B was purchased from GE Healthcare (Australia). Standard dextrans, ascorbic acid, EDTA, ferrozine, Dimethyl Sulfoxide (DMSO) were purchased from Sigma–Aldrich (Australia). Penicillin, streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), Lipopolysaccharides (LPS), foetal bovine serum (FBS) and glutaMAXTM were purchased from Life Technologies (Mulgrave, Australia). Mouse RAW 264.7 macrophages (ATCC number TIB-71) were purchased from American Type Culture Collection (ATCC). All other reagents were of analytical grade. 2.2. Extraction and fractionation of polysaccharides from Lupinus angustifolius The seeds of L. angustifolius were powdered with a kitchen blender (Sunbeam PB7600). The powder is then subjected to autoclaving with water (for 2 h at 121 ◦ C), then cooled to laboratory temperature and then filtered with a filter paper. The supernatant was then treated with 95% ethanol (1:4 vol/vol) for about 15 h at 4 ◦ C. Crude polysaccharides were obtained by centrifugation (10,000 × g for 20 min) and then were dissolved in distilled water.

The sample was then freeze-dried to obtain dry polysaccharide extract and was stored at −20 ◦ C until further studies (Jeong et al., 2012). Size-exclusion chromatography (SEC) was then performed using a custom packed column (packed with Sepharose CL-6B; 2.4 × 99 cm). The column was equilibrated with distilled water and 3 mL of polysaccharide extract was injected into the column (at a concentration of 10 mg/mL) and eluted with distilled water at a flow rate of 0.42 mL/min to obtain molecular weight fractionation. Phenol–sulfuric acid method was then employed to obtain the sugar profile of the eluted fractions (Masuko et al., 2005; Jeong et al., 2012). Six polysaccharide fractions were collected as showed in Fig. 1 (Section 3.1). These fractions resulting from 25 SEC runs have been pooled and lyophilized. 2.3. Estimation of average molecular weight Average molecular weights of the polysaccharide fractions were estimated by calibrating the SEC column using a series of standard dextran samples (2000 KDa, 450 KDa, 150 KDa, 70 KDa, 40 KDa, 10 KDa) and Glucose. The calibration data has provided a standard linear regression (y = 0.0005x + 0.0943) with a high correlation coefficient (R2 = 0.923) which was used to estimate the apparent average molecular weights of extracted blue lupin polysaccharide fractions (Jeong et al., 2012; Zhang et al., 2013). 2.4. Determination of total sugar content and mono-sugar composition of polysaccharide fractions In order to estimate the total sugar contents phenol–sulfuric acid method (Masuko et al., 2005) has been used. The monosugar contents of isolated polysaccharides were analysed using a Hewlett Packard 7890B gas chromatograph (Agilent Technologies, CA, USA) fitted with a FID detector. A HP-5 capillary column (Agilent Technologies, CA, USA) with 30 m × 0.32 mm I.D. and 0.25 ␮m film thickness was used separate and detect the mono-sugars. Acid hydrolysis (using trichloroacetic acid) method was employed to produce mono-sugars from polysaccharide fractions and the resulting mono-sugars were acetylated (Zhang et al., 2013) before injecting into the gas chromatograph for analysis. Mono-sugar standards (fucose, mannose, xylose, glucose, arbinose, galoctose, rhamnose and ribose) were also acetylated and used for gas chromatographic analysis (Zhang et al., 2013) 2.5. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra have been recorded with PerkinElmer Spectrum 100 Spectrometer equipped with universal ATR accessory.

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This accessory enables quick FT-IR analysis of solid/powder samples without the need for sample preparation. All the FT-IR spectra were recorded with a scan range of 4000–650 cm−1 , a frequency resolution of 1 cm−1 and 32 scans. All recordings were baseline corrected. 2.6. Antioxidant activities of polysaccharide fractions 2.6.1. ABTS+• radical scavenging activity ABTS+• radical scavenging assay was used to determine the antioxidant properties of lupin polysaccharides (Kao and Chen, 2006). The procedure involved, mixing of ABTS diammonium salt (0.35 mL at 7.4 mmol/L) with potassium persulfate (0.35 mL at 2.6 mmol/L) to obtain ABTS+• radical. In order to allow complete radical formation, the mixture was kept at room temperature in a dark room for 15 h with aluminium foil rapped around. To obtain the absorbance reading of 0.70 ± 0.02 at 734 nm, the radical solution was diluted with 95% ethanol (about 1:40 vol/vol). The scavenging activity was measured by adding 2 mL of ABTS+• solution 0.2 mL of polysaccharide fractions (95% ethanol was used as negative control). The absorbance was measured at 734 nm at 20 min after the initial mixing (95% ethanol was used as the blank). The scavenging activity was calculated as: Scavenging activity (%) =



1−



A × 100 A0

where, A0 is the absorbance at 734 nm of 95% ethanol (negative control) and A is the absorbance at the same wave length of the polysaccharide sample. 2.6.2. Ferrous chelating activity The chelating activity of polysaccharide fractions was measured by monitoring their abilities to complex with ferrous ions in the presence of ferrozine. Eleven samples of each polysaccharide fraction were prepared with different concentrations (0–5 mg/mL). Each one of these samples were mixed with 0.1 mL of 2 mM iron(II) chloride, 0.15 mL of 5 mM ferrozine and 0.55 mL of MeOH. The samples were thoroughly mixed and allowed to react at room temperature for 10 min. Spectrophotometer was then used to measure the absorbance of the samples at 562 nm. The ability of EDTA to form complex was used as positive control (Kao and Chen, 2006). Percentage chelating activity was calculated as follows: Chelating activity (%) =



1−

A A0



× 100

Where, A0 is the absorbance measured with EDTA as positive control at 562 nm and A is the absorbance measured with polysaccharide samples at the same wavelength. 2.7. Griess assay Polysaccharide samples with different concentrations were prepared (0.3–10 mg/mL) for testing their ability to stimulate RAW 264.7 macrophages. 96-well microtiter plate was used in this assay with a cell density of 2 × 105 cells per well. Macrophage cells were incubated in the medium containing 20 ␮L of polysaccharide samples at 37 ◦ C (in 5% CO2 ) for 24 h. 0.1 mL of the supernatant was collected from the culture and mixed with 0.1 mL of Griess reagent (consisting of 0.1% [wt/vol] naphthyl ethylenediamine and 1% [wt/vol] sulfanilamide in 5% [vol/vol] phosphoric acid). Samples were kept at room temperature for 20 min and then NO production was monitored using sodium nitrite as a standard by colorimetric method (Cui et al., 1994; Jeong et al., 2012). LPS was used as positive control.

2.8. Cell viability assay Cell viability of RAW 264.7 macrophages was tested with MTT assay (Dore et al., 2013). Cell viability was measured with different concentrations (0.3–10 mg/mL) of lupin polysaccharide fractions. Briefly, the cells were incubated for 24 h at 37 ◦ C in a 96-well microtitre plate as described in section 2.7 and the supernatant was removed. Then, 100 ␮L of DMEM medium containing MTT (0.2 mg/mL) was added to each well and the microtitre plate containing the cells was further incubated for 2 h at 37 ◦ C. The supernatant was removed and 50 ␮L of DMSO was added to each well. The plate was shaken for 30 min to solubilize the formazan crystals and absorbance was measured at 595 nm. LPS was used as positive control. Following equation was used to calculate the cell viability (%): Cell viability (%) =



1−

A A0



× 100

where, o is the measured absorbance with positive control (LPS) at 595 nm and is the absorbance at the same wavelength with polysaccharide samples. 2.9. Data analysis Data analysis was done using SPSS Software (Version 20, IBM SPSS, Chicago, IL, USA) and Microsoft Excel. All the data was collected in triplicate and the results were expressed as mean ± standard deviation (SD) values. The group mean was compared using a one-way analysis of variance and Duncan’s multiple range tests. The data were considered statistically significant if pvalue was <0.05 (significant at the 5% level). A small p-value means that the observed parameter is highly significant. 3. Results and discussion 3.1. Extraction and fractionation of polysaccharides from Lupinus angustifolius L. (blue lupin) Crude polysaccharides were extracted from powdered seeds of L. angustifolius by autoclaving (section 2.2). A yield of 99.6 g of polysaccharide extract per kilogram of lupin seeds was obtained. The total carbohydrate content of this extract was 54.63% (the protein content was 44.36%). Sepharose CL-6B SEC column was employed to obtain molecular weight fractionation of blue lupin polysaccharides. Six fractions were collected based on their carbohydrate elution profile (Jeong et al., 2012; Zhang et al., 2013). These separated polysaccharide fractions are designated as BLP-1, BLP-2, BLP-3, BLP-4, BLP-5 and BLP-6 (Fig. 1). Size exclusion fractionation of 1 g of polysaccharide extract yielded 62.4 mg of BLP-1, 129.6 mg of BLP-2, 232.3 mg of BLP-3, 307.2 mg of BLP-4, 108.8 mg of BLP5 and 96.3 mg of BLP-6. Antioxidant activities of BLP-3 and BLP-4 have been observed to be low and hence these fractions are not considered any further in this paper. The apparent average molecular weights of isolated polysaccharide fractions were estimated as described in Section 2.3 and are presented in Table 1. It should be noted that the average molecular masses estimated by this method are not accurate (Jeong et al., 2012). However, they are of great help to understand the influence of the molecular size on biological activities. 3.2. FT-IR spectroscopic characterisation of active polysaccharides Figs. 2 and 3 present FT-IR spectra of four of the active blue lupin polysaccharide fractions isolated in this study and it is interesting

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% Transmittance

100

7

90 8 1

80 2 5 6

4000

3500

3000

2500

2000

Wavenumbers

4

1500

3

1000

(cm-1)

Fig. 2. FT-IR spectrum of BLP-6. Relevant vibrational bands in the spectrum are identified with numerals 1–8 (Band-1: 877.9 cm−1 ; Band-2: 989.5 cm−1 ; Band-3: 1067.5 cm−1 ; Band-4: 1103.3 cm−1 ; Band-5: 1539.4 cm−1 ; Band-6: 1640.5 cm−1 ; Band-7: 2931.8 cm−1 and Band-8: 3277.3 cm−1 ).

Fig. 3. FT-IR spectra of BLP-1, BLP-2 and BLP-5. Vibrational bands and their positions in these spectra are identical to those represented in Fig. 2.

to see that all of them have similar infrared absorption bands indicating similarities in their structural features. The FT-IR spectrum of BLP-6 (Fig. 2) has a peak at 877.9 cm−1 indicating the presence of ␤-glycosidic linkages (Pawar and Lalitha, 2014). The three absorption peaks at 989.5 cm−1 , 1067.5 cm−1 and 1103.3 cm−1 correspond to the stretching vibrations of C O in C O H bonds of pyranose ring confirming that BLP-6 contains pyranose sugars (Pawar and Lalitha, 2014). The broad band centred around 3277.3 cm−1 corresponds to the hydroxyl stretching vibrations of the polysaccharide and the peak at 2931.8 cm−1 belongs to C H stretching vibrations. The band at 1640.5 cm−1 corresponds to the stretching vibration

Table 1 Carbohydrate and protein contents, molecular masses and mono-saccharide compositions of polysaccharide fractions isolated from blue lupin.

Average molecular mass (kDa) Carbohydrate content (%) 0 Protein content (%) Monosaccharide (%) Galactose (%) Fucose (%) Glucose (%) Mannose (%) Rhamnose (%) Xylose (%) Ribose (%) Unknown (%) a

BLP-1

BLP-2

BLP-5

BLP-6

2301 79.15 20.84

1882 50.13 49.86

46 54.09 45.90

3 24.37 75.62

79.42 20.57 – – – – – –

45.39 24 5.23 10.7 8.12 2.93 3.59

48.73 16.41 13.84 8.03 10.52 2.44 – –

19.89 8.49 21.14 7.32 8.82 2.17 3.63 28.54a

–

One of the mono-sugar present in BLP-6 was not included in the standards used in this study. Hence, it was not possible to identify.

of the carbonyl bond that is a part of amide group and the band at 1539.4 cm−1 is related to the N H bending vibration of the same group. Occurrence of these two vibrations due to amide group indicates the presence of protein. These observations confirm that the blue lupin polysaccharide structures contain pyranose sugars with ˇ-glycosidic linkages. Further studies to evaluate the detailed structures of blue lupin polysaccharides are underway in our laboratory.

3.3. Monosaccharide compositions of the fractions Mono-sugar compositions of the four polysaccharide fractions have been analysed as described in section 2.5 and these results are presented in Table 1. It can be noted from these results that, most of the fractions consist mainly of galactose, fucose, rhamnose, glucose and mannose. They also contain small quantities of ribose and xylose. It is important to note that most of the polysaccharide fractions (BLP-1, BLP-2, BLP-5 and BLP-6) isolated from blue lupin contained large proportion of galactose (Table 1) and average levels of glucose and mannose. These observations suggest that the polysaccharides derived from blue lupin may contain galactomannans, glucomannans, mannans and galactans. Possible occurrence of galactomannans in blue lupin is consistent with the literature that endosperms of the seeds mainly contain galactomannans (e.g. Leguminosae) (Tester and Al-Ghazzewi, 2013). It is interesting to note from the literature that galactomannans have several pharmaceutical/industrial applications (Cerqueira et al., 2011; Tester and Al-Ghazzewi, 2013; Srivastava and Kapoor, 2005).

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ABTS radical scavenging avvity (%)

70 60

(a)

50 40 30 20 10 0

3.4. ABTS+• radical scavenging ability of polysaccharide fractions ABTS+• radical scavenging abilities of blue lupin polysaccharide fractions are presented in Figs. 4 and 5 their IC50 values are presented in Table 2. Three of the polysaccharide fractions (BLP1, BLP-2 and BLP-5) exhibited significant scavenging capacity in a dose dependant manner (Figs. 4 and 5). Excellent IC50 values (Table 2) were observed for BLP-1, BLP-2 and BLP-5. Interestingly, these three active fractions contain large quantity of galactose (Table 1). Moderate scavenging activity was displayed by BLP-6 and this fraction has moderate quantity of galactose (Table 1). BLP3 and BLP-4 displayed lowest scavenging activity and contained low levels of galactose (results not presented). These observations indicate that galactose is likely to play a key role in the antioxidant activities of blue lupin polysaccharides. Other monosaccharides present in moderate quantities in active fractions are fucose, glucose, mannose and rhamnose. These findings strongly indicate the correlation between ABTS+• scavenging activity of lupin polysaccharide fractions and their mono-sugar content. For example, the fractions containing large quantity of galactose displayed highest activity indicating that the antioxidant activities of blue lupin polysaccharides have direct correlation to their galactose content. Contribution of other mono-sugars (such as glucose, mannose and rhamnose) to radical scavenging activity is also evident. It is interesting to note from the literature that mung bean polysaccharides display significant radical scavenging activities and mainly contain mannose, galactose and rhamnose (Lai et al., 2010; Zhong et al., 2012). Combination of literature observations and the findings of this paper points to the possible contribution of galactomannans, glucomannans and galactans to the observed antioxidant activities of blue lupin derived polysaccharides (Section 3.4). However, further research is required to establish this hypothesis.

Table 2 Antioxidant activities of polysaccharide fractions isolated from blue lupin. Data are presented as the mean ± SD (n = 3). Polysaccharide fractions

IC50 value (mg/mL) (ABTS scavenging activity)

BLP-1 BLP-2 BLP-5 BLP-6

6.98 7.68 5.34 8.45

± ± ± ±

0.72 1.12 0.91 0.83

IC50 value (mg/mL) (Ferrous chelating activity) 0.40 1.52 0.55 1.91

± ± ± ±

0.69 0.95 0.72 0.61

2 4 6 8 10 Concentraon of polysaccharide fracons (mg/mL)

12

60 50

(b)

40 30 20 10 0 0

2

4

6

8

10

12

Concentraon of polysaccharide fracons (mg/mL)

ABTS radical scavenging avvity (%)

Fig. 4. ABTS radical scavenging activities of four polysaccharide fractions isolated from blue lupin. (ABTS radical scavenging activities are expressed in percentage with respect to the scavenging ability of Vitamin C at 0.25 mg/mL taken as 100%) (n = 3, p < 0.05).

ABTS radical scavenging avvity (%)

0

80 70

(c)

60 50 40 30 20 10 0 0

2

4

6

8

10

12

Concentraon of polysaccharide fracons (mg/mL) Fig. 5. Dose dependant ABTS radical scavenging activities of three of the blue lupin polysaccharide fractions: (a) BLP-1, (b) BLP-2 and (c) BLP-5. (ABTS radical scavenging activities are expressed in percentage with respect to the scavenging ability of Vitamin C at a concentration of 0.25 mg/mL taken as 100%) (n = 3, p < 0.05).

3.5. Ferrous chelating and reducing power of lupin polysaccharide fractions Ferrous chelating activity of blue lupin polysaccharides have been determined as described in section 2.6 and the results are presented in Table 2 and Fig. 6. These results indicate that four of the blue lupin polysaccharide fractions display significant ferrous chelating activity. BLP-1 and BLP-5 have displayed highest metal chelating ability. It is interesting to note that BLP-1 and BLP-5 exhibited high radical scavenging activity and also high chelating activity. It is pertinent to note here that radical scavenging activity and ferrous chelating activity are both important characteristics of a potential antioxidants (Perron and Brumaghim, 2009). Presence of excessive free radicals in a biological system can lead to the process of DNA damage and hence can cause serious diseases including

120

955

200

100 180

80 60 40 20 0 0

1

2

3

4

5

6

Concentraon of polysaccharide fracons (mg/mL)

NO production (%)

Ferrous chelang acvity (%)

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160 MΦ

140

BLP-1 BLP-2

120

BLP-5 BLP-6

100

Fig. 6. Dose dependant ferrous chelating activities of the blue lupin polysaccharide fraction: BLP-5. (Ferrous chelating activities are expressed in percentage with respect to the chelating ability of EDTA at a concentration of 0.562 mg/mL taken as 100%) (n = 3, p < 0.03).

80 0

1

2

3

4

5

Concentration of polysaccharide fractions (mg/mL) cancer. Hence, radical scavenging activity is an essential process by which oxidative free radicals can be removed and DNA damage can be prevented (Perron and Brumaghim, 2009). Also, the presence of excessive ferrous ions in a biological system can also lead to DNA damage. Ferrous chelating ability of polysaccharides is therefore expected to decrease the presence of free Fe2+ ions and hence eliminates this mechanism of DNA damage. Blue lupin polysaccharides possess both radical scavenging and chelating activities. Hence, they have strong potential to be used as natural antioxidants. The tremendous health benefits of lupin kernel fibre already known in the literature (Belski, 2012) together with the observed antioxidant activities of lupin derived polysaccharides strongly points to their beneficial utilisation. Blue lupin polysaccharides are therefore strong candidates for nutraceutical applications. Minimal toxicity (Fig. 7) of these polysaccharides is an added advantage for such applications. It is pertinent to mention here that the antioxidant activities of blue lupin polysaccharides are not as high as herbal polysaccharides (Zhang et al., 2013). However, considering the fact that these are food polysaccharides and can be consumed in larger quantities make them beneficial for human health. 3.6. Immunostimulatory activity of lupin polysaccharides The influence of blue lupin polysaccharides on the cell viabilities are presented in Fig. 7. It is interesting to note that all the polysaccharide fractions exhibited excellent cell viabilities even at high concentration (10 mg/mL) (Fig. 7) and hence are non-toxic.

BLP-1

90

BLP-2

Cell viability (%)

85

BLP-5

Fig. 8. Effect of blue lupin polysaccharides on RAW 264.7 macrophage activation leading to nitric oxide (NO) production (n = 3, p < 0.05).

Literature demonstrates that botanic polysaccharides exhibit least toxicity (Schepetkin and Quinn, 2006; Jeong et al., 2012) and the present results are consistent with these observations. Lupin polysaccharides significantly stimulated the macrophages as monitored by measuring the production of nitric oxide (Fig. 8). Immunostimulatory effects (NO production) were measured by stimulating mouse macrophages (RAW264.7) with lupin polysaccharide fractions. Dose dependant increase of nitric oxide (NO) production was observed indicating the ability of lupin polysaccharides to activate these cells (Fig. 8). All fractions induced the production of NO by more than 160% when compared to untreated macrophages (100%). BLP-2 displayed highest activity and BLP1 and BLP-3 showed significant activity (Fig. 8). These findings indicate that blue lupin polysaccharides are suitable candidates to stimulate the macrophage function and improve immune system. Literature demonstrates that polysaccharides derived from legumes such as guar gum contain galactomannans and display significant immunomodulatory activity (Naito et al., 2006). It is also reported in the literature (Tester and Al-Ghazzewi, 2013) that mannose containing polysaccharides display immunomodulatory and anti-cancer activities in addition to their many other biological activities. Most of the lupin polysaccharides studied here contained significant quantities of galactose and mannose which may be responsible for their immunostimulatory activities. Literature indicates that antioxidant and immunostimulatory activity plays an important role in anti-cancer activity (Luo and Fang, 2008; Zhang et al., 2013). It is therefore expected that the polysaccharides isolated from blue lupin are likely candidates for anti-cancer activities.

BLP-6

80

4. Conclusion

75 70 65 60 55 50 0

2

4

6

8

10

12

Concentration of polysaccharide fractions (mg/mL) Fig. 7. Cell viabilities of polysaccharide fractions from Lupinus angustifolius (blue lupin) (n = 3, p < 0.05).

Lupin polysaccharides have been successfully extracted, fractionated and their antioxidant and immunostimulatory activities have been determined in this study. They displayed significant radical scavenging, iron chelating and immunostimulatory activities. Antioxidant activities of these polysaccharides showed considerable relationship to their mono-saccharide composition. Direct correlation was found between the radical scavenging activities and their galactose content. Other mono-sugars that are likely to be responsible for antioxidant activities are mannose, rhamnose and glucose. Outcomes of this research strongly indicate that blue lupin polysaccharides have great industrial potential to be used as nutraceuticals/therapeutic agents with least toxicity. FT-IR inves-

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