Selected Sri Lankan food plants and other herbs as potential sources of inulin-type fructans

The objective of this study was to determine the inulin-type fructan content in 20 selected food plants and other herbs commonly found in Sri Lanka. The inulin content of the selected plants were determined qualitatively and quantitatively using thin layer chromatography (TLC) and enzymatic spectrophotometric (ES) methods, respectively. The ES results showed that the inulin-type fructan contents based on fresh weight was highest in Allium sativum (18.62 % ± 1.55), followed by Asparagus falcatus (17.74 % ± 2.92), Asparagus racemosus (11.8 3% ± 0.87), Allium cepa (8.60 % ± 0.88), Allium ampeloprasum (6.20 % ± 0.23), Taraxacum javanicum (5.77 % ± 1.53) and Vernonia cinerea (4.55 % ± 0.93), respectively. Taraxacum javanicum and Vernonia cinerea plant extracts developed distinct blue black spots with the detection reagent on TLC plates similar to chicory inulin standard. However, Allium ampeloprasum, Allium cepa, Allium sativum, Asparagus falcatus and Asparagus racemosus developed thicker blue black streaks on TLC plates due to their higher inulin concentration, which confirmed the ES results. Aloe vera, Alpinia calcarata, Amophophallus campanulatus, Beta vulgaris, Canna indica, Diascorea alata and Sonchus oleraceus FW) of inulin while Caryota urens, Ipomoea batatas, Lasia spinosa and Maranta arundinacea contained very low levels or no (< 0.4 g/100 g FW) inulin.


INTRODUCTION
Inulin-type fructans are non digestible, polydisperse carbohydrate materials made up of fructose units, fructose linkages with an optional terminating glucose molecule. They are considered linear or branched fructose polymers with a degree of polymerization of 2-60 (Roberfroid, 2005;2007b).
Inulin-type fructans have gained much interest in the food industry as 'functional food ingredients' because they have the ability to selectively stimulate the activities and growth of beneficial microflora (specifically Bifidobacteria and some Lactobacillus species) in the human colon, maintain a healthy balance of microflora in the colon and promote the integrity of colon epithelium (Roberfroid, 2005). They are well documented and proven as 'prebiotics', and have been claimed to improve intestine immune functions, reduce blood cholesterol levels, improve absorption of Ca and Mg, reduce the risk of irritable bowel diseases (IBD) and more importantly reduce the risk of colon cancer (Sako & Tanaka, 2002).
Inulin-type fructans naturally occur as storage carbohydrates in some families of plants representing approximately 30,000 species (Niness, 1999;Singh & Singh, 2010). Plants in the families Asteraceae, Asparagaceae, Amaryllidaceae and Campanulaceae are well known to store inulin-type fructans. Several studies have shown the distribution of inulin-type fructans in a variety of food plants (Loo et al., 1995;Campbell et al., 1997;Judprasong et al., 2011;Moongngarm et al., 2011). There are many food plants and herbs in Sri Lanka, which may contain high levels of inulin-type fructans.

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Journal of the National Science Foundation of Sri Lanka 43 (1) Currently widely marketed commercial inulin is extracted from two plants of the family Asteraceae; Cichorium intybus (chicory) and Helianthas tuberosus (Jerusalem artichoke), which are not found in Sri Lanka. Commercial inulin extracted from chicory is imported to Sri Lanka from European countries at a high cost for food processing applications. Therefore it is important to search for locally available sources of inulin-type fructans for commercial inulin extraction. Hence this study was aimed at screening selected food plants and other herbs in Sri Lanka for inulin-type fructans to identify potential plant sources for inulin extraction.

Selection of plants
The selection of plants was based on previous studies on inulin-type fructan contents in food plants (Loo et al., 1995;Campbell et al., 1997;Judprasong et al., 2011) and medicinal plants used in Ayurveda, the Sri Lankan traditional medicine and the alternative medicine for treating digestive tract related disorders (Jayasinghe et al., 1979;1985;Jayaweera, 1980;1981a;b;1982

Extraction of fructan for TLC analysis
The edible portions of the cleaned and peeled parts of the plants were weighed (6 g) and cut into small pieces of 2 mm, and immediately introduced into conical flasks filled with 50 mL of warm (70 C) distilled water. The conical flasks were covered with parafilm and placed on a shaking water bath at a constant temperature (85 ± 2 C) for 15 min to extract the fructans and sugars (McCleary et al., 2000).

Preparation of standards and detection reagent
Glucose, fructose and sucrose standards were prepared at a concentration of 3 mg/mL. Potato starch (Thomas Baker Chemicals, India), chicory inulin and chicory fructooligo saccharide (FOS) standards (Beno Orafti, Belgium) were prepared at a concentration of 5 mg/mL. The detection reagent was prepared by dissolving diphenylamine (2 g) and aniline (2 mL) in acetone (80 mL), carefully adding 15 mL of phosphoric acid and diluting to 100 mL with acetone. 100 mL of the detection reagent was used as dipping solution (Reiffova & Nemcova, 2006).

Qualitative thin layer chromatography (TLC) analysis
Thin layer chromatography analyses of the plant extracts were carried out as described by Reiffova and Nemcova (2006) with slight modifications. Prior to TLC analysis, chromatographic silica gel 60 F 254 (200 × 200 mm) pre-coated aluminum sheets (Merck KGaA, Germany) were pre-treated with 0.02 M sodium acetate, followed by drying in an oven at 50 C for 5 min. Equal volumes (approximately 0.5 µL) of glucose, fructose, sucrose, potato starch, inulin, FOS standards and plant extracts were spotted manually on TLC plates using capillary tubes. The TLC plates were developed two times in butanol:ethanol:water [5:3:2 (v/v/v)] solvent system at room temperature with air drying in between the two developments. After development, the plates were dried for 10 min in a stream of warm air. The plates were then dipped in detection reagent for 5s and allowed to dry at room temperature for 10 min. Blue-black spots were visualized by heating the plates in an oven at 120 C for 20 min.

Enzymatic spectrophotometric method
The fructan contents in the plant extracts were determined using the enzymatic spectrophotometric method (AOAC method 999.03) using Megazme fructan assay kit (Megazyme, Ireland), which contained sucrase, fructanase, fructan control, sucrose control and D-fructose standard.

Extraction of fructan for enzymatic spectrophotometric method
Cleaned (peeled) plant parts were grated using a cheese grater (thickness < 1 mm), accurately weighed (1.0 g) and immediately put into conical flasks filled with 80 mL of hot distilled water (~70 C). The fructans and sugars were extracted as explained earlier in TLC analysis. The extract was allowed to cool to room temperature and quantitatively transferred into a 100 mL volumetric flask. The volume was adjusted to 100 mL with distilled water, thoroughly mixed and filtered through a Whatman No. 1 filter paper. The filtered solution was used for analysis immediately. Fructan control powder (25.5 % fructan) (Megazyme, Ireland) was extracted (100 mg in 50 mL of distilled water) using the same procedure along with the samples (Megazyme, 2012).

Preparation of enzymes and reagents
Sucrase/ amylase and fructanase enzyme solutions were prepared as described by McCleary et al. (2000) and stored in a freezer at -20 C until use. D-fructose standard solution and para-hydroxybenzoic acid hydrazide (PAHBAH) reducing sugar assay reagent

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Journal of the National Science Foundation of Sri Lanka 43 (1) were prepared as described in McCleary et al. (2000), immediately before use.

Removal of sucrose, starch and reducing sugars
Aliquots of the extracted sample (0.2 mL) was mixed with diluted sucrase/ amylase enzyme solution (0.2 mL) in a glass test tube and incubated at 40 C for 30 min. Then 0.2 mL of freshly prepared alkaline borohydrate solution (10 mg/mL sodium borohydride in 50 mM sodium hydroxide) (Sigma-aldrich, USA) was added to the sample, vigorously stirred and incubated at 40 C for 30 min. The samples were then mixed with 0.5 mL of 0.2 M acetic acid followed by vigorous stirring on a vortex mixer. This extract solution was used for hydrolysis and measurement of fructans (McCleary et al., 2000).  ., 2000).

Measurement of fructans
PAHBAH working reagent (5.0 mL) was added to all test tubes (samples, sample blanks, reagent blanks, fructan control, D-fructose standard) and immediately immersed in a boiling water bath for exactly 6 min (McCleary et al., 2000). All the tubes were then placed in cold water within 10 -15 min against reagent blank at 410 nm using UV-visible spectrophotometer (UV-1601, Shimadzu Co., Japan). Three independent fructan extractions and measurements were performed for each plant species used in the study. In order to validate the method of analysis, standard inulin-type fructan samples (25.5 % inulin) were analysed in parallel to each independent measurement.

Proximate moisture, ash, crude fat, crude protein and crude fiber contents
Moisture, ash, fat, protein and fiber content of the plant samples were determined according to the methods described in AOAC (2005).

Total water soluble sugars
Total water soluble sugars of the plant samples were determined using the phenol-sulfuric method using glucose as the standard (Dubois et al., 1956). Approximately 1.0 g of pre-prepared plant sample was weighed and extracted in 100 mL of hot water as described under TLC analysis, and 1.0 mL aliquot of each plant extract was diluted in 250 mL volumetric flasks using distilled water. An aliquot (1.0 mL) of the diluted solution was transferred to a glass test tube (16 × 100 mm) and mixed with an equal volume (1.0 mL) of 5 % (v/v) phenol. Concentrated sulfuric acid (5.0 mL) was added to each test tube, mixed well and allowed to incubate at 30 C in a water bath. The absorbance was measured at 490 nm using UV-visible spectrophotometer (UV-1601, Shimadzu Co., Japan).
The Megazyme fructan assay results were in high level of agreement with the results of TLC analysis (Figure 1) in which Asparagus falcatus (13), Allium sativum (17) and Asparagus racemosus (28) showed thick blue-black streaks on the TLC plates due to high fructan concentrations in the sample. Allium cepa (18) and Allium ampeloprasum (26) developed rather thin blue-black streaks on TLC plates, while Vernonia ceneria (19) and Taraxacum javanicum (25) (medium to low fructan sources) showed distinct spot patterns similar to chicory inulin standard (Figure 1).

Scientific name
Inulin-type fructan content (g/100 g fresh basis)     The inulin level in Allium ampeloprasum (leeks) in this study (6.2 ± 0.23 g/100 g FW) was in agreement with that reported by Loo et al while Allium cepa showed a higher inulin content (8.6 ± 0.88 g/100 g FW) than that was reported by Loo et al. in The inulin level in Asparagus racemosus found in this study (11.83 ± 0.87 g/100 g) was in accordance with (1997).
The differences in the reported values may be due to the analytical method used, the variation in climatic conditions, soil conditions, maturity levels and varieties of the particular plant (Carvalho et al., 1998;Kociss et al., 2007;Vandoorne et al., 2012). Loo et al. (1995) reported Taraxacum officinale FW). However, Taraxacum javanicum found in Sri Lanka showed only 5.77 ± 1.53 g/100 g FW inulin.
According to the present study Maranta arundinacea (Arrowroot), which is commonly used in Ayurveda and Sri Lankan traditional medicine for correcting bowel complaints and diarrhea (Jayasinghe et al., 1979;Jayaweera, 1982) ) contained only 0.04 ± 0.01 g/100 g FW of inulin, indicating that most probably its medicinal effect is delivered through another mechanism.

Some other constituents of plant samples
The results of the physico-chemical analysis are presented in Table 4. The moisture content was lowest in Caryota urens pith flour (13.66 g/100 g), followed by Maranta arundinacea (61.97 g/100 g) and Diascorea alata (63.41 g/100 g). As expected, the moisture content was highest in Aloe vera leaf gel (95.68 g/100 g).

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Journal of the National Science Foundation of Sri Lanka 43 (1) Allium species showed the highest crude protein values among the analyzed plants, namely Allium sativum (19.20 g/100 g), Allium ampeloprasum (15.71 g/100 g) and Allium cepa (12.99 g/100 g) on dry weight basis.
The crude fiber content on dry matter basis was the highest in Vernonia cinerea (13.73 g/100 g) followed by Lasia spinosa (13.34 g/100 g) and Taraxacum javanicum (12.9 g/100 g). Canna indica (green), Allium sativum and Allium cepa showed comparatively high crude fat contents among the analyzed plants; 4.67, 4.63 and 4.33 g/100 g, respectively on dry matter basis.
According to the present study, the inulin content in Asparagus falcatus (17.74 ± 1.29 g/100 g FW) was comparable to that of chicory (Cichorium intybus), which was reported as (Loo et al., 1995). Thus Asparagus falcatus could be a potential source for the commercial production of inulin.