Extraction, purification and characterization of a new Water-soluble polysaccharide from Acanthophyllum bracteatum roots Kambiz jahanbina*, Sohrab Moinib, Ahmad Reza Goharib, Zahra Emam-Djomeha a
Department of Food Science and Engineering, Faculty of Biosystem Engineering, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran (
[email protected]) b Faculty of Pharmacy, Medicinal Plants Research Center, Medical Sciences/University of Tehran, Tehran, Iran (
[email protected])
ABSTRACT Recently there has been an increase in the demand of polysaccharides. Polysaccharides from plants have the advantage over those from animals because of their friendly image towards consumers. A lot of work has been done to study the chemical composition and functionality of polysaccharides. However, there is still place in the polysaccharide market for new sources of plant polysaccharides. Acanthophyllum C. A. Meyer is a genus belongs to the Caryophyllaceae family, with a total of 61 species in the world. Of these 33 occur in Iran of which 23 species are endemic. The highest number of species has been recorded in east of Iran (Khorrasan province) and in the adjacent area i.e., Turkmenistan and Afghanistan. We have examined the Water-soluble polysaccharide from the roots of Acanthophyllum bracteatum (one of the most important species from this genus). The polysaccharide extracted in different conditions (alkaline and acid) and the alkaline extraction yield (14%) was higher than the acid extraction yield (9%). The polysaccharide preparation had a specific optical rotation of 170◦, moisture content of 8.3% and ash content of 0.6%. After purification and acid Hydrolysis the carbohydrate composition was analyzed by HPLC that revealed the presence of galactose, glucose and arabinose in a ratio of 19.66, 8.66 and 1.00, respectively and negligible amounts of rhamnose and galacturonic acid. The results indicated that the polysaccharide of the roots was of the arabinoglucogalactan type. This is the first report on isolation of a polysaccharide from Acanthophyllum bracteatum. Keywords: Extraction; polysaccharide; Acanthophyllum bracteatum; Chemical composition
INTRODUCTION The genus Acanthophyllum (Caryophyllaceae) is represented by 61 species in the world of which 23 species are endemic in Iran [1]. According to literature the highest number of species has been recorded in the east of Iran (Khorasan province) and in adjacent area i.e., Turkmenistan and Afghanistan [2]. The east of Afghanistan towards China and the west of Turkey towards Syria are poor in species, where only a single species occurs in China (A. punges ) and Syria (A. verticillatum). Considering the floristic regions, all the species of this genus belong to Irano-Turanian region [3]. They are popularly known as ‘‘chubak’’ and used as a detergent in the Khorasan province. Their roots also were used as an expectorant, emetic and vulnerary for horses. Acanthophyllum bracteatum is one of the most important species from this genus. There are few reports on the chemical constituents of Acanthophyllum species, except that they contain saponins, polysaccharides and monosaccharides [4-7]. MATERIALS & METHODS 2.1. Chemicals and Plant materials The roots of A. bracteatum were collected by the authors and stored at room temperature. Taxonomic identification was done by Prof. Valiollah Mozaffarian, Botanical taxonomist. Pure monosaccharide standards of D-mannose (Man), L-rhamnose (Rha), D-ribose (Rib), D-galactose (Gal), D-xylose (Xyl), Darabinose (Ara), D-glucose (Glu) and L-galacturonic acid were obtained from Merck Co. and Sigma Chemical Co. (St. Louis, MO, USA). Aqueous solutions were prepared with ultra-pure water from a Milli-Q water purification system (Millipore, Bedford, MA, USA). All other reagents used were of analytical grade.
2.2. Extraction of polysaccharide The polysaccharide extraction followed a procedure described by Arifkhodzhaev [4] with some modifications. Briefly, Samples weighing 100 g of hypogeal organs of the plant were first inactivated three times with chloroform in a ratio of 1:10 in the boiling water bath for 2 h, and, after filtration, the plant residue was dried in the air and it was then inactivated twice with methanol in a ratio of 1:10 at the boil for 2 h and, after filtration, the plant material was dried. The raw material after inactivation was extracted with water in a ratio of 1:6 at room temperature and two different pH (4, 10) for 2 h. Extraction was repeated twice more and the extracts were combined. The protein was eliminated by Sevag's method [8] and the aqueous solution was evaporated in vacuum rotary evaporator at 40°C to a volume of 0.5 liter. Polysaccharides were precipitated with alcohol in a ratio of 1:3. The precipitate was separated off by centrifugation, washed with alcohol, and water was removed by freeze-drying (Christ Alpha 2-4 freeze-drier). 2.3. Determination of moisture content The crucible and its cover were dried in an oven for 30 min at 100 ºC. Then, the crucible was cooled desiccator and was weight without cover to four decimal points. Approximately 2 g of polysaccharide weighed in the crucible and it was placed in the oven for 6 h at 100 ºC. After drying, the dried sample then covered and cooled in a desiccator. Later, the crucible was weighed without cover. Moisture measured in the form of weight decrease after heating the sample.
in a was was was
2.4. Determination of ash content The total ash content was determined according to AOAC Official Method 923.03. [9]. About 5 g of polysaccharide was weighed into a shallow, relatively broad ashing dish that has been ignited, cooled in desiccator and weighed soon after reaching room temperature. Then, the sample was ignited in a furnace at 600 ºC for 4 h. The ash content was measured as the residue remaining after heating at 600 ºC for 4 h. 2.5. Determination of optical rotation Optical rotations were measured at 20 ºC using a Perkin–Elmer 343 polarimeter. 2.6. Determination of sugar composition Monosaccharides composition was determined by HPLC. The HPLC system used had a K-1001 Pump (Knauer, Germany) equipped with a 20 μL sample loop linked with a EUROKAT H column (8 × 300 mm) (Knauer, Germany) and a refractive index detector. Data evaluation was performed using the peak area and an external standard. 0.25 ml of HCL 2M added to 4 mg of sample and the tube of sample were heated for 5 h in a water bath at 100°C. The solutions were neutralized by adding barium carbonate to obtain pH=7 and then the hydrolysates were filtered (0.45 μm) and analyzed by HPLC. RESULTS & DISCUSSION The polysaccharide yield obtained by alkaline extraction (14%) was higher than that from acid treatment (9%). This result is probably because of more hydrolysis in acid extraction that causes some loss of the polysaccharide. The Acanthophyllum bracteatum polysaccharide had a moisture content of 8.3% and ash content of 0.6% (Table 1). Specific optical rotation [α] for Acanthophyllum bracteatum polysaccharide was 170 (Table 1). This result explains that the glycose probably has a high number of α-linkages.
Table 1. Summary of physicochemical Composition of Acanthophyllum bracteatum polysaccharide.
parameter moisture ash optical rotation
value 8.3% 0.6% 170
Results obtained from HPLC (Table 2) revealed the presence of galactose, glucose and arabinose as major components and minor quantities of rhamnose and galacturonic acid. These results confirm an arabinoglucogalactan as the main polysaccharide in Acanthophyllum bracteatum root. The sugar composition of polysaccharide extracted from Acanthophyllum bracteatum root is similar to that of other polysaccharides from the Acanthophyllum genus [4-7]. The ratio of galactose, glucose and arabinose was 19.66, 8.66 and 1.00, respectively. Table 2. Monosaccharides composition of Acanthophyllum bracteatum polysaccharide (Standard errors are shown between parentheses).
Monosaccaride galactose glucose arabinose rhamnose galacturonic acid
% (w/w) 66 (0.8) 29 (0.5) 3.3 (0.1) 0.7 (0.02) 0.9 (0.02)
The results indicated that the polysaccharide of the roots of Acanthophyllum bracteatum was of the arabinoglucogalactan type and this genus can be use as a new source of polysaccharide. CONCLUSION The polysaccharide extracted in different conditions (alkaline and acid) and the alkaline extraction yield (14%) was higher than the acid extraction yield (9%). The polysaccharide preparation had a specific optical rotation of 170◦, moisture content of 8.3% and ash content of 0.6%. After purification and acid Hydrolysis the carbohydrate composition was analyzed by HPLC that revealed the presence of galactose, glucose and arabinose in a ratio of 19.66, 8.66 and 1.00, respectively and negligible amounts of rhamnose and galacturonic acid. The results indicated that the polysaccharide of the roots was of the arabinoglucogalactan type.
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