Scavenging and antioxidant activities of immunomodulating polysaccharides isolated from Salvia officinalis L.

International Journal of Biological Macromolecules 44 (2009) 75–80

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Scavenging and antioxidant activities of immunomodulating polysaccharides isolated from Salvia officinalis L. P. Capek ∗ , E. Machová, J. Turjan Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia

a r t i c l e

i n f o

Article history: Received 19 September 2008 Received in revised form 16 October 2008 Accepted 17 October 2008 Available online 30 October 2008 Keywords: Sage Polysaccharides Antioxidant activity Scavenging ability Structural analysis

a b s t r a c t Crude polysaccharides, isolated from the aerial parts of sage (Salvia officinalis L.) by sequential extraction with water (A), hot ammonium oxalate (B), dimethyl sulfoxide (C), 1 M (D) and 4 M (E) potassium hydroxide solutions, and six ion-exchange fractions of A were examined for their ability to inhibit peroxidation of liposome lipid by hydroxyl radicals and to reduced DPPH• radical content. The highest inhibition of liposome lipid peroxidation was found with crude polysaccharides A, B and D, antioxidant activities reached ∼37%. The purified fractions A1 and A2 inhibited the liposome peroxidation to ∼35%. However, the radical scavenging abilities of the most active crude polysaccharides A, B and C on DPPH• radicals were found in the range 80–90%, while the most active purified fractions A3 –A6 in three or fourfold doses achieved 75–92%. The least effective tested polysaccharides succeeded 20% inhibition using both methods. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Medicinal plants represent a diverse group of herbs spread throughout the world with a high content of bioactive compounds possessing a variety of biological activities. Of them, polysaccharides or their glycoconjugates were shown to exhibit multiple biological activities including anticarcinogenic, anticoagulant, immunostimulating, antioxidant, etc. Presently much attention has been focused to the antioxidant effect of various microbial or plant natural compounds inclusive of carbohydrate polymers because they become more and more important due to their large application in food or pharmaceutical industries. Generally, antioxidants represent a diverse group of hydrophilic and hydrophobic types of natural compounds playing important role in the protection of living organisms. The elimination of a negative role of radicals which are created during a cascade of oxido-reduction reactions, by UV radiation, toxic compounds, heavy metals, etc. in cells, plays an important role in the defence of cellular components or organelles, i.e. lipids, proteins, glycoconjugates, nucleic acids, etc. It has been found that free radicals play a key role related to the degenerative or pathological processes of various diseases, such as cancer, Alzheimer’s disease, coronary heart disease, atherosclerosis, neurodegenerative disorders, aging, cataracts, and various

∗ Corresponding author. Tel.: +421 2 59410209; fax: +421 2 59410222. E-mail address: [email protected] (P. Capek). 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.10.007

inflammations [1–7]. Studies concerning the antioxidant and/or scavenger activities on crude plant polysaccharide extracts, purified polysaccharides or glycoproteins from various medicinal plants indicate that these plant carbohydrates showed significant antioxidant activities and can be explored as potential antioxidants [8–13]. Consequently, searching for active natural compounds with possible antioxidant and/or radical scavenger properties, has still a great importance. Until now, no investigation has been carried out on sage polysaccharides concerning scavenging and/or antioxidant activities. Therefore, in this paper, the immunomodulatory active crude polysaccharides obtained by sequencial extraction of aerial part of sage and purified polysaccharide fractions of water-extractable polysaccharide complex A [14,15] were investigated for their possible radical scavenging and/or antioxidant abilities by DPPH• radical assay and hydroxyl radicals, generated via Fenton’s reaction initiated by hydrogen peroxide and ferric chloride. 2. Materials and methods 2.1. Plant material and chemicals Dried aerial parts of medicinal plant (Salvia officinalis L.) were obtained from Slovakofarma (Malacky, Slovakia). DPPH• (1,1-diphenyl-2-picrylhydrazyl) radical, DPPH (1,1-diphenyl-2picrylhydrazine), ␣-Tocopherol and DEAE-Sephacel were purchased from Sigma–Aldrich (Germany).

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2.2. General methods

2.4. DPPH radical scavenging assay

Polysaccharides were hydrolyzed with 2 M trifluoroacetic acid for 1 h at 120 ◦ C. Concentration of solutions were performed under diminished pressure at a bath temperature below 40 ◦ C. The quantitative determination of the neutral sugars was carried out in the form of their trifluoroacetates by gas chromatography on a Hewlett-Packard 5890 Series II chromatograph using a PAS-1701 column (0.32 mm × 25 m) at a temperature program of 110–125 ◦ C (2 ◦ C/min)−165 ◦ C (20 ◦ C/min) and a flow rate of hydrogen 20 mL/min [16]. The uronic acid content was determined with the 3-hydroxybiphenyl reagent [17]. The methoxyl group content was determined according to the Vieböck–Schwappach method in a Zeisel apparatus [18]. Determination of molecular mass was performed with Shimadzu apparatus (Vienna, Austria) using a tandem of two columns HEMA-BIO 100 followed HEMA-BIO 40 column (Tessek, Prague, Czech Republic) of dimensions 8 mm × 250 mm. As a mobile phase 0.02 M phosphate buffer pH 7.2 containing 0.1 M NaCl was used at a flow rate 0.8 mL/min. A set of pullulan standards was used for calibration of the column (Gearing Scientific, Polymer Lab. Ltd., UK). Fourier transform infrared spectroscopy (FT-IR) of samples were recorded with a Nicolet Magna 750 spectrometer with DTGS detector and OMNIC 3.2 software. The polysaccharides were pressed into KBr pellet with a sample/KBr ratio 1/200 mg.

Free radical scavenging ability of crude sage polysaccharides A–D and purified polysaccharides A1–A6 against DPPH• (2,2diphenyl-2-picrylhydrazyl) radical was determined spectrophotometrically [20]. Three different concentrations of polysaccharides in distilled water (0.008 wt%, 0.016 wt% and 0.032 wt%) were tested. To 1 mL of polysaccharide sample (0.08, 0.16 and 0.32 mg/mL) a solution (0.08 mg/1 mL) of DPPH• radical (0.008 wt% solution in methanol) was added and allowed to react at laboratory temperature for 1 h after mixing with vertex. When DPPH• radicals are reduced the changes in colour (from violet to yellow) were measured at 517 nm on a SPECORD M-20 (Zeiss, Jena, Germany) UV–vis spectrophotometer against a blank. Analyses were performed in triplicate. Free radical scavenging ability (FRSA) was calculated as follows: FRSA (%) = (Ao − Atest )/(Ao − Aref ) × 100, where Ao was the absorbance of free DPPH• radicals, Atest was the absorbance of DPPH• radicals treated with sample for 1 h, and Aref was the absorbance of DPPH solution (0.008% solution in methanol). Results were expressed as a mean ± standard error of the mean (S.E.M.) from three independent experiments.

2.3. Isolation of sage polysaccharides Dry aerial parts of medicinal plant (S. officinalis L.) were cut into small pieces and extracted under reflux with methanol–chloroform (10:1 v/v) to remove coloured extractive compounds. The drug residue was further macerated twice in distilled water for 24 h at laboratory temperature in the presence of 0.02% solution of natrium azide. Both aqueous extracts were concentrated, combined and precipitated with ethanol (1:4 v/v) containing 1% of acetic acid. The precipitate was dissolved in distilled water, dialyzed and freezedried to give the crude polysaccharide A. The residue was further macerated twice in 0.5% aqueous ammonium oxalate for 2 h at 90 ◦ C. The combined solutions were concentrated, dialyzed and freeze-dried to give the crude polysaccharide B. The depectinated residue was slightly washed with distilled water and macerated twice in dimethyl sulfoxide for 24 h at laboratory temperature. Solutions were combined, exhaustively dialyzed, concentrated and freeze-dried (C). The remaining residue washed with water and treated twice with 1 M aqueous potassium hydroxide solution containing 10 mM solution of NaBH4 for 2 h at laboratory temperature. The alkaline extracts were cooled, neutralized with 4 M acetic acid, concentrated, dialyzed, and freeze-dried to give the hemicellulose fraction D. The plant residue was finally treated twice with 4 M aqueous potassium hydroxide containing 10 mM solution of NaBH4 for 2 h at laboratory temperature. The extracts were recovered in the same way as above to give the hemicellulose fraction E. The crude polysaccharide A was dissolved in distilled water and applied to a column of DEAE-Sephacel and eluted with water (A1), 0.1 M (A2), 0.25 M (A3), 0.5 M (A4) and 1 M (A5) sodium chloride solutions, and finally with 1 M sodium hydroxide solution (A6). Fractions of 10 mL were collected and analyzed for the carbohydrate content by phenol–sulfuric acid assay [19]. The fractions eluted with sodium chloride solutions were concentrated, dialyzed and freeze-dried to give the polysaccharides A1–A5. The coloured fraction eluted with 1 M sodium hydroxide solution was neutralized, concentrated, dialyzed and freeze-dried to give the brownish polysaccharide A6.

2.5. Liposome preparation Multilamellar liposomes were prepared by the hydration method. 15 mg of lipid soybean phosphatidylcholine (Sigma, St. Louis, USA) was dissolved in 1 mL of the mixture of chloroform–methanol (2:1 v/v). The lipid solution was evaporated in vacuum by a rotary evaporator. The residual lipid film was hydrated with 3 mL of distilled water (pure liposomes) or aqueous solutions of the polysaccharides to be tested. The hydration of lipid film was realized on reciprocal shaker (10 strokes/min). The final lipid concentration was 5 mg/mL in all the experiments. The concentration of polysaccharides tested was from 0.01 to 0.2 wt%. 2.6. Induction of lipid peroxidation Natural phospholipids contain only nonconjugated double bonds and absorb the light at very short wavelength ( = 200–220 nm). Removal of a hydrogen atom from a methylene group located between two double bonds spreads the unsaturation over five carbon atoms and results in the formation of a conjugated diene which is energetically more favourable than the two double bonds in isolation. As a result, the second absorbance maximum at 233 nm appears [21]. For the study of lipid peroxidation of liposomes we have used OH• radicals produced via Fenton’s reaction initiated by addition of H2 O2 and FeCl2 with a final concentration of 100 mM and 2 mM, respectively, to the liposome suspension containing varying concentrations of polysaccharides tested. To 1 mL liposomes (pure or with the incorporated tested polysaccharides), 0.2 mL Fenton reagent (6.7 mg FeCl2 , 120 ␮L H2 O2 , 1.88 mL H2 O) was added and allowed to react for 30 min. Then 0.1 mL sample was withdrawn, 2 mL ethanol was added and after 15 min the absorption spectra of conjugated dienes were measured in the wavelength region of 190–270 nm. 2.7. Determination of oxidation index Absorption spectra of conjugated dienes were recorded in the wavelength range 215–320 nm using a SPECORD M-20 (Zeiss, Jena, Germany) UV–vis spectrophotometer. The increase in the absorption at 233 nm was considered as an evidence of the formation of conjugated dienes, and the oxidation index was calculated from the ratio of the absorbances (A233 /A215 ) [22,23]. The antioxidant activity (AOA) was expressed as AOA (%) = 100 × (IL − IC )/IL , where IL and

P. Capek et al. / International Journal of Biological Macromolecules 44 (2009) 75–80 Table 1 Sugar composition, protein content and molecular size distribution of crude polysaccharides A–E extracted from aerial parts of sage. Sugar composition (mol%)

A

B

C

D

E

Rhamnose Fucose Arabinose Xylose Mannose Glucose Galactose 3-O-methyl-Galactose Uronic acid (mol%) Protein (wt%) Mw × 103

6.7 2.6 30.4 7.6 8.3 15.5 17.9 3.0 8.0 9.4 2–93

3.2 Tr. 13.4 1.5 2.2 4.4 6.6 – 68.6 9.0 100

4.5 Tr. 32.5 6.6 5.1 17.1 23.6 – 10.7 7.5 10, 80

1.5 – 12.0 66.6 – 5.6 6.7 – 7.6 19.0 4–5

3.0 – 15.4 35.0 8.3 18.1 10.7 – 9.7 10.6 4–5

Tr.: trace.

IC stand for the Klein index of the pure liposome and the tested compound, respectively. Results were expressed as a mean ± standard error of the mean (S.E.M.) from three independent experiments. 3. Results 3.1. Isolation and structural characterisation of crude sage polysaccharides Decoloured and defatted aerial parts of sage were gradually extracted with cold water (A), hot aqueous ammonium oxalate (B), dimethyl sulfoxide (C), 1 M (D) and 4 M (E) potassium hydroxide solutions. From water macerate the crude polysaccharide (A) was obtained by ethanol precipitation, followed by dialysis and freeze-drying. Polysaccharides from ammonium oxalate (B) and dimethyl sulfoxide (C) solutions were recovered by dialysis and freeze-drying. Polysaccharides extracted by alkaline solutions (D and E) were recovered by neutralization, followed by dialysis and freeze-drying. Extractions of aerial parts of sage with water, ammonium oxalate, dimethyl sulfoxide, 1 and 4M potassium hydroxide solutions yielded approximatly 4%, 8%, 1%, 5% and 6% of polysaccharides (on dry weight basis), respectively. The crude polysaccharide A displayed a broad molecular-mass distribution pattern with Mw 2000–93,000. Monosaccharide analysis showed a dominance of arabinose, galactose and glucose residues, followed by uronic acid, mannose, xylose and rhamnose (Table 1). Structural studies on A revealed a highly branched ␣-Larabino-␤-3,6-D-galactan consisting of 1,6-linked galactopyranose backbone carrying ␣-L-arabinosyl side chains and methoxyl groups at C-3 [24], as its dominant component (over 50 wt%), followed by glucomannan with a backbone composed of ␤-1,4-linked glucoand mannopyranosyl units slightly branched at C-6 by side chains, mainly as single ␣-glucosyl and mannosyl stubs [25], which represents about 14 wt% of A. Rhamnose and uronic acids (∼14–15 mol%) indicate the presence of the acidic type of polymers, i.e. galacturonan and/or rhamnogalacturonan in A. Besides, it contains 9.4 wt% of protein which in plant materials are usually linked with arabinogalactans. The polysaccharide material B, extracted by hot ammonium oxalate, showed only one peak at Mw ∼ 100,000. Compositional analysis revealed a dominance of uronic acids (∼70 mol%), arabinose and galactose (∼20 mol%), and lower amounts of glucose, rhamnose, mannose and xylose residues (∼10 mol%) indicating the pectic type of polymers in B. Their presence was also confirmed by the bands on FT-IR spectrum (not shown) at 1606 (␯as COO− ) and 1417 cm−1 (␯s COO− ), and at 1101, 1077, 1047, and 1018 cm−1 [26]. Besides, very low rhamnose content (∼3 mol%) indicates the prevalence of homogalacturonan content in this material (Table 1).

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Polysaccharides C, solubilized by dimethyl sulfoxide, showed a broad molecular-mass distribution pattern with Mw 2000–100,000. The dominant components were arabinose and galactose (∼60 mol%), followed by glucose, uronic acid, xylose, mannose and rhamnose. It indicates the presence of arabinogalactan as the dominant component, xyloglucan and pectic material. The rhamnose/uronic acid ratio 1:2.4 suggests the prevalence of highly rhamnified type of rhamnogalacturonan in C (Table 1). Besides, low contents of accompanying coloured components, proteins and phenolic compounds (in IR spectrum the band at 1512 cm−1 ) were identified. Hemicellulose polysaccharides D, solubilized by 1 M potassium hydroxide, showed a high degree of polydispersity with one distinct peak only at Mw ∼ 50,000. On hydrolysis yielded a high proportion of xylose (67 mol%) and arabinose residues (12 mol%) residues, while other sugars were found in low amounts. The presence of xylan and/or glucuronoxylan-related polysaccharides was confirmed by the main bands on FT-IR spectrum at 1047 cm−1 and at 897 cm−1 suggesting the ␤-type of glycosidic linkage [27]. Moreover, in this material were determined phenolic compounds and the highest protein content (∼19 wt%) of all fractions. Hemicellulose polysaccharides E, extracted by 4 M potassium hydroxide, showed a broad molecular-mass distribution pattern with three maxima at Mw ∼ 10,000; 12,000 and 70,000 with a dominance of last one. Monosaccharide analysis revealed the lower content of xylose residues (35 mol%) in comparison with D (67 mol%), while galactose, glucose and mannose residues significantly increased which indicates the presence of another hemicellulose polysaccharide, a galactoglucomannan in E (Table 1). The FT-IR spectrum of E showed band maxima at 1047 and 897 cm−1 confirming the presence of xylan-related polysaccharides and phenolic component at 1517 cm−1 . A lower content of proteins (∼10–11 wt%) in comparison with D was determined.

3.2. Ion-exchange chromatography of the polysaccharide complex A and characterization of its fractions The water-extractable polysaccharide complex A is substantial from the point of view of therapeutic applications. To obtain any indication of the presence of polysaccharide types constituting A, it was further resolved by ion-exchange chromatography into six fractions by step-wise elution with water (A1), sodium chloride solutions (A2–A5) and sodium hydroxide (A6). The fractions eluted with water and sodium chloride solutions were concentrated, dialyzed and polysaccharide fractions A1–A5 were recovered by freeze-drying. The strongly bounded fraction eluted with 1 M sodium hydroxide solution was neutralized, concentrated, dialyzed and freeze-dried to give brown polysaccharide material (A6). The polysaccharide fraction A1 showed one symmetrical peak with Mw ∼ 8000. The dominant sugar components were arabinose, galactose, glucose and mannose residues while uronic acids, xylose, rhamnose, and fucose were present in smaller proportions (Table 2). Two polysaccharides, i.e. a highly branched ␣-L-arabino␤-3,6-D-galactan and a linear ␤-1,4-linked glucomannan have been identified in A1 as the main components [24,25]. The polysaccharide fraction A2 displayed one dominant peak at Mw ∼ 11,000 and one smaller peak at Mw ∼ 60,000. Sugar analysis revealed two main components, i.e. arabinose and galactose (∼75 mol%) and indicated an arabinogalactan as the main polysaccharide in this fraction, similarly as in A1 (Table 2). Its presence was confirmed by the bands on FT-IR spectrum at 1073 and 1037 cm−1 belonging to arabinogalactan moiety [27]. Moreover, uronic acids (∼9 mol%) and rhamnose (∼5 mol%) suggest a rhamnogalacturonan type of polymer in A2 as the minority component.

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Table 2 Sugar composition and molecular size distribution of ion-exchange fractions A1–A6 of water extracted polysaccharide complex A. Sugar composition (mol%)

A1

A2

A3

Rhamnose Fucose Arabinose Xylose Mannose Glucose Galactose 3-O-methyl-Galactose Uronic acid (mol%) Mw × 103

1.2 0.4 35.0 2.9 10.1 13.0 22.1 10.6 4.7 8

4.8 Tr. 34.4 1.5 4.2 6.0 23.4 17.1 8.6 11, 66

11.8 0.7 32.5 2.0 5.6 6.7 21.8 – 18.9 2, 9, 66

A4 18.7 2.0 25.5 1.7 3.3 6.6 18.3 – 23.8 3, 13, 120

A5

A6

8.9 2.0 34.3 2.5 8.0 14.3 23.8 – 6.2 5

6.8 2.4 37.7 2.3 8.6 16.2 21.2 – 4.8 5

Tr.: trace.

The polysaccharide material A3 showed a broad molecular-mass distribution pattern with three maxima at Mw ∼ 2000; 9000 (a dominant component) and 66,000. The sugar composition analysis revealed the predominance of galactose, arabinose, uronic acids, and rhamnose (Table 2) and indicated the presence of arabinogalactan–rhamnogalacturonan complexes which represent 85 mol% of all sugar constituents. The FT-IR spectrum showed characteristic bands maxima at 1073 and 1037 cm−1 for arabinogalactan and at 1734, 1612, 1414, 983, 917, 900, and 820 cm−1 for rhamnogalacturonan moieties [27]. Polysaccharides of A4 assigned to molecular heterogeneity with two main components at Mw ∼ 3000 and 13,000 and minority one at Mw ∼ 120,000, similarly as in fraction A3. On hydrolysis sample yielded arabinose, uronic acids, rhamnose, and galactose as the dominant sugar components while the other sugars were detected only in low amounts. A4 contained the highest proportions of uronic acids and rhamnose residues (43 mol%) of all ion-exchange fractions. The low rhamnose/uronic acid ratio 1:1.3 suggests the presence of highly rhamnified type of rhamnogalacturonans which can be associated with arabinogalactans as indicate the high proportion of arabinose and galactose residues (44 mol%) in Table 2. The polysaccharide fraction A5, eluted with 1 M sodium chloride solution, showed one symmetrical peak at Mw ∼ 5000. It was rich mainly in arabinose, galactose and glucose residues (Table 2). Relatively high content of mannose (8 mol%) and rhamnose (9 mol%) residues was observed while uronic acids content markedly decreased (∼6 mol%). It seems that arabinogalactans associated with phenolic compounds were main components of this material. Their presence was confirmed in FT-IR spectrum by the bands at 1072 and 1036 cm−1 and at 1512 cm−1 [28]. Dark brown polysaccharide material A6, eluted with 1 M sodium hydroxide, showed similar molecular-mass distribution pattern as that of A5, i.e. one symmetrical peak at Mw ∼ 5000. The dominant components were arabinogalactans (∼60 wt%), as indicates sugar composition in Table 2, associated with phenolic compounds (bands at 1512 cm−1 in FT-IR spectrum). Relatively high content of mannose (∼9 mol%) and glucose (∼16 mol%) residues could indicate the presence of glucomannan as a minority component in A6.

rides A–E, measured in the concentration range of 0.008–0.032% (0.08–0.32 mg/mL) using the DPPH colorimetric assay, are shown in Fig. 1. As it can be seen, all polysaccharides except that of A, showed in all concentrations dose-dependent DPPH• radical scavenging activities. Besides, the scavenging activities of polysaccharides B and C increased very significantly with increasing concentrations. The results from in vitro experiments demonstrated that the crude polysaccharide B had the highest radical scavenging ability (∼90%), followed by polysaccharide C (∼82%), and water fraction A (∼78%) at a dose of 0.32 mg/mL. Polysaccharides D and E showed the lowest radical scavenging activity of all crude fractions (∼20%). However, in the lower doses (0.08 and 0.16 mg/mL), the polysaccharide A showed the highest radical scavenging activity of all crude polysaccharides and its activity increased significantly with increasing concentrations only in the range 0.08 and 0.16 mg/mL, while in concentrations 0.16 and 0.32 mg/mL no significant differences were found. Relatively high effect of A can be connected with the main structural components, i.e. arabinogalactans (∼50 wt%), glucomannans (∼15 wt%) and pectic type of polymers-rhamnogalacturonans (∼15 wt%), or their glycoconjugates as indicates the protein content (∼9 wt%). It has been found that uronic acid and especially galacturonic acid or its polymer, i.e. polygalacturonic acid showed very strong antioxidant activity in vitro and some correlation between molecular size and activity was observed [29]. However, the polysaccharide A contains 8 mol% of uronic acid and consequently these negative charged molecules cannot be the only reason of this high effect. As it can be seen from Fig. 1, pectic polymers of B at the dose of 0.16 mg/mL, showed much lower effect in comparison with complex A, although contained about 70 mol% of

3.3. The free radical scavenging of sage polysaccharides For investigation of the radical scavenger abilities of sage polysaccharides, the DPPH• free radical scavenging assay was used. This assay is routinely used for monitoring of the scavenging abilities of many natural compounds. When DPPH• radicals are reduced in the model system with an antioxidant compound, the changes in colour from violet to yellow are measured at 517 nm. The scavenging abilities of crude and purified water-extractable sage polysaccharides against DPPH• radicals were investigated. The radical scavenging activities of the crude sage polysaccha-

Fig. 1. Scavenging effects of crude sage polysaccharides A–E.

P. Capek et al. / International Journal of Biological Macromolecules 44 (2009) 75–80

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Fig. 2. Scavenging effects of purified sage polysaccharides A1–A6.

Fig. 3. Antioxidant activities of crude sage polysaccharides A–E on peroxidation of soybean lecithin liposomes.

uronic acids. In order to find the most active component in A, it was resolved into six distinct fractions A1–A6 by ion-exchange chromatography. Individual polysaccharide fractions A1–A6, measured in the same concentration range, showed lower radical scavenging activities than did crude polysaccharides A–C (data not shown). However, their effects increased with increasing concentration of polysaccharides to 0.032–0.1 wt% (0.32–1.0 mg/mL). As it is shown in Fig. 2, the polysaccharide fractions A1–A4 demonstrated in all concentrations dose-dependent DPPH• radical scavenging activities, while effects of A5 and A6 fractions were similar in the doses 0.64–1.0 mg/mL. Of the purified fractions, the highest radical scavenging abilities were found in A3 and A6 (over 90%), followed by polysaccharides A5 (over 80%) and A4 (70%). Polysaccharides A1 and A2 showed very low activities (∼20%). Their detail structural analyses revealed the presence of arabinogalactans (70–75 wt%) and glucomannans (10–15 wt%), and low content of rhamnogalacturonans. Moreover, both neutral polymers showed relatively low average molecular mass (8000–11,000). It is evident that neutral polymers of A1 and A2 do not assist significantly to the total activity of A. However, A3 and A4 fractions, except arabinogalactans contain the highest proportion of rhamnogalacturonans of all purified fractions, and it seems that they can significantly contribute to the total activity of A. Similarly, low molecular mass polymers of A5 and A6, rich in arbinogalactans and hexosans significantly contribute to the total activity of A probably due to the low content of phenolic compounds. As it can be seen from Fig. 1, the most active components of sage polysaccharides are pectic polymers B. Their structural analysis revealed a high content of galacturonic acid and indicated thus the prevalence of homogalacturonan type of polymer in this fraction. The low rhamnose content can be an integral part of slightly rhamnified rhamnogalacturonan to which neutral arabinogalactan side chains can be attached. Besides, analytical data revealed as well the presence of proteins (∼9 wt%), acetyl and methoxyl groups and indicated the presence of partly esterified pectic macromolecules. Polysaccharides C, solubilized by dimethyl sulfoxide were shown to be important from the point of view of their high scavenging activity. Arabinogalactans were found as the dominant component, followed by rhamnogalacturonans, glucomannan and some xylan-derived polymers could be identified. Besides, colour compounds, proteins and appearance of low content of phenolic compounds could indicate the existence of synergistic effect of these minorite ingredients.

Surprising were relatively low activities of D and E polymers in which xylan and/or glucuronoxylan-related polysaccharides were dominant components. Although, in both fractions the highest contents of colour compounds, proteins and phenolic compounds have been determined of all tested samples, no synergistic effect of these accompanying ingredients has been observed. 3.4. The antioxidant activities of sage polysaccharides The antioxidant activities of crude and purified sage polysaccharides against the peroxidation of soybean lecithin liposomes induced by • OH radicals generated in Fenton’s reaction were investigated. The crude polysaccharides A–E used in the concentration range of 0.01–0.2 wt% (0.1–2.0 mg/mL) exhibited antioxidant activities up to ∼40% (Fig. 3). It has been demonstrated that only polysaccharides B and C showed the dose-dependent effect in all used concentrations. Although, the antioxidant activity of C increased more expressively with increasing concentrations in comparison with polysaccharide B, it was less active. The pectic polysaccharide B is one of the most active component of sage and even at the dose 0.1 mg/mL its antioxidant activity was the most expressive (∼27%). In the case of polysaccharides A, D and E, their activities significantly increased only in the doses 0.1–1.0 mg/mL, while in higher one (2.0 mg/mL) lower effects were observed. Moreover, in the dose 1.0 mg/mL, polysaccharides A and D were shown to exhibit the highest antioxidant activities (∼37%) of all the tested samples. To this value only the polysaccharide B is approximating, however, at much higher dose, i.e. 2.0 mg/mL. As seen from the Fig. 3, all crude polysaccharides, except the hemicellulose E, exhibited relatively high antioxidant activity ranged from 30% to 37%. It is evident, that crude sage polysaccharides with the synergistic effect of minor accompanying components are able to protect biological membranes of liposomes and thus prevent their peroxidation. The ion-exchange fractions of A, i.e. A1–A6, measured in the same concentration range, exhibited the antioxidant activity as well (data not shown), however, their effect was lower in comparison with the crude polysaccharide complex A. The higher doses, i.e. 1.0 and 5.0 mg/mL increased significantly their effects. As seen from Fig. 4, the polysaccharides A1, A2 and A5 showed the dosedependent effect, while in other fractions, i.e. A3, A4 and A6 the lower effects of higher doses (5.0 mg/mL) have been noticed. Of all purified fractions, the highest antioxidant activities were deter-

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Acknowledgements This work was supported by the Slovak Scientific Grant Agency (VEGA), Grant No. 2/0155/08 and the Science and Technology Assistance Agency (APVV), Grant No. 0030-08. The authors are grateful to E. Marková for technical assistance. References

Fig. 4. Antioxidant activities of purified sage polysaccharides A1–A6 on peroxidation of soybean lecithin liposomes.

mined in A2 (∼35%), A3 (∼28%), and A4 (∼27%). The activities of other purified fractions, i.e. A4–A6 were lower and succeeded about 20%. Besides, in the dose 1.0 mg/mL, the polysaccharide A3 was found to be the strongest inhibitor of liposome peroxidation (∼28%) of all purified samples. However, the fivefold dose revealed remarkable increase in the inhibition of liposome peroxidation only in the fraction A2. Its value (∼35%) was very close to the value of the most active water-extractable polysaccharide complex A (∼37%). The results of this study revealed the important role of crude and purified sage polysaccharides in the protection of liposomes as experimental biological membranes to the free radical attack. It has been found that sage polysaccharides, crude or purified were strong scavengers of free radicals as well as inhibitors of liposomes peroxidation. Their antioxidant activities were higher than effects of polysaccharides isolated from medicinal plants Arctium lappa var. herkules, Aloe barbadensis, Althaea officinalis var. robusta, Plantago lanceolata var. libor, Rudbeckia fulgida var. Sullivantii or Mahonia aquifolium described in our previous paper [9].

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