Lamiaceae carbohydrates. 1. Pectinic substances and hemicelluloses from Mentha x piperita

Article abstract of DOI:10.1007/s10600-007-0177-z

Pectinic substances from the aerial part of Mentha x piperita were isolated and characterized and found to be a mixture of β-(1→4)-glucogalactan (MPG) and two α-(1→4)-rhamnopolygalacturonans (MPP'-1 and MPP'-2). It was shown that the pectin and its components exhibited membrane-stabilizing, antiatherogenic, and antioxidant activity. Hemicelluloses from M. piperita were a lignocarbohydrate complex.

Full text of DOI:10.1007/s10600-007-0177-z

Chemistry of Natural Compounds, Vol. 43, No. 5, 2007
LAMIACEAE CARBOHYDRATES. 1. PECTINIC SUBSTANCES
AND HEMICELLULOSES FROM Mentha x piperita
D. N. Olennikov and L. M. Tankhaeva
UDC 582.949.27+547.917
Pectinic substances from the aerial part of Mentha x piperita were isolated and characterized and found to
be a mixture of β-(1→4)-glucogalactan (MPG) and two α-(1→4)-rhamnopolygalacturonans (MPP′-1 and
MPP′-2). It was shown that the pectin and its components exhibited membrane-stabilizing, antiatherogenic,
and antioxidant activity. Hemicelluloses from M. piperita were a lignocarbohydrate complex.
Key words: Mentha x piperita, Lamiaceae, pectinic substances, glucogalactan, rhamnopolygalacturonan,
hemicelluloses, biological activity.
Mentha x piperita L. (Lamiaceae) is an official plant material that is widely used in medical practice. The main
properties of pectinic substances (PS) from the variety Krasnodar 116 [1] and polysaccharides from tissue culture of domestic
[
2] and foreign [3, 4] varities of this species have been previously described.
Herein we report results from the isolation and study of the composition of PS and hemicellulose (HC) from the aerial
part of M. piperita Krasnodar 2 variety.
Wefound 5.23-6.13% PS containing 97.02±2.01%carbohydratesand<0.02%phenoliccompoundswith 0.85±0.16%
ash content, 0.52 ± 0.02% nitrogen, [α]D20 +189° (c 0.1, water), and pH 5.5-5.8 (c 0.1, water) in the aerial part ofM. x piperita.
The GalUA content was 54.5% with the ratio of neutral carbohydrates in the total complex PS being
Glc:Gal:Ara:Rha: Xyl = 28:25:5:4:1 (Table 1). The quantitative characteristics were free carboxylic group content (KC),
5
.35 ± 0.11%; esterified carboxylic groups (K ), 4.39 ± 0.14%; esterification degree (λ), 45-46%.
E
PS were separated using calcium pectinate in order to determine the component composition. This produced acidic
and neutral components, MPP and MPG, respectively. Saponification bybase of MPP gave demethoxylated pectinic acid MPP′,
which was confirmed by analyzing the functional groups: K = 9.48 ± 0.28%; K = 0.34 ± 0.08%; λ = 3-4%.
C
E
Gel chromatography showed that MPP′ consisted of two components MPP′-1 and MPP′-2 in the ratio 1:2.3 with
4
4
MW 6.0·10 and 3.7·10 Da, respectively. The monosaccharide compositions of these were identical and differed only by the
lack ofXyl in MPP′-2. Partial hydrolysisofMPP′-1andMPP′-2formedthesamechromatographicallyhomogeneous component
MPP′′, which contained 97.3% GalUA and 2.7% Rha. The molecular weight of MPP′′ according to gel chromatography was
4
3
.0·10 Da; the degree of polymerization, 185.
Enzymatic hydrolysis of MPP′′ by pectinase produced GalUA. The positive specific rotation of MPP′′ indicated that
the glycoside bonds had the α-configuration. This was confirmed by the insignificant decrease of the specific rotation of the
polyacetate (MPP′′-Ac) compared with starting MPP′′. The content of acetyls corresponded to substitution of two hydroxyls in
anhydrogalacturonyl units. TheIRspectrum ofMPP′′ containedthefollowingabsorption bands(cm 1): 1725 (carboxylicC=O);
–
1
9
610 and 1400 (ionized carboxylic); 1028, 1050, 1110 (pyran ring), and 832 (α-glycoside bond). The bands at 740 and
22 cm – shifted compared with pure GalUA (775 and 904 cm –1). This is characteristic of polysaccharides with a 1→4 bond.
Methoxylated polygalacturonan waspreparedand subjected toperiodate oxidation in order toprove the type ofbonding
1
in MPP′′. The product from oxidation of methoxylated polygalacturonan was levorotary and reduced a solution of silver in
ammonia, i.e., acted like a polyaldehyde. The hydrolysate contained traces of GalUA, which eliminated a 1→3 bond from
consideration. Oxidation by nitric acid of P produced oxalic and tartaric acids (1→4 bond). Oxidation by CrO gave a
al
3
hydrolysate in which GalUA and traces of Rha (α-bond) were detected.
Institute of General and Experimental Biology, Siberian Division, Russian Academy of Sciences, 670047, Ulan-Ude,
ul. Sakh′yanovoi, 6, fax (3012) 43 30 34, e-mail: oldaniil@rambler.ru. Translated from Khimiya Prirodnykh Soedinenii,
No. 5, pp. 411-416, September-October, 2007. Original article submitted January 10, 2007.
©
0
009-3130/07/4305-0501 2007 Springer Science+Business Media, Inc.
501

TABLE 1. Quantitative Monosaccharide Composition of PS and Its Components and Periodate Oxidation Results, mol %
HCOOH
Fraction
Ara
Gal
Glc
Rha
Xyl
GalUA
NaIO4*
isolation*
PS
MPP
3.8
1.7
1.9
2.2
1.9
-
17.8
14.8
10.7
13.7
11.8
-
19.7
10.2
10.0
11.9
5.8
-
3.1
1.7
1.9
1.4
2.1
2.7
6.5
-
0.7
0.6
1.7
8.8
-
-
0.8
-
54.5
71.0
73.7
53.4
78.3
97.3
-
-
-
-
-
-
-
MPP′
MPP′-1
MPP′-2
MPP′′
MPG
MPG′
0.92
0.94
1.02
0.97
1.01
0.324
0.250
0.016
0.305
0.030
9.8
-
48.8
100
34.2
-
-
_
_____
*
mol/anhydrous unit.
Oxidation of MPP′′ by NaIO consumed 1.02 mol/anhydrous unit (1→4 hexapyranose). The amount of HCOOH was
4
0
.016 mol/anhydrous unit, which indicated a linear or slightly branched structure for the macromolecule with the given degree
of MPP′′ polymerization. The position of Rha in the MPP′′ structure was established by methylation with diazomethane and
reduction with NaBH followed by Hakomori methylation of the reduced product until the hydroxyl absorption band in the
4
IR spectrum disappeared. TLC of the hydrolysate from the permethylate after formolysis detected 2,3,6-tri-O-Me-Gal; 2,3,4,6-
tetra-O-Me-Gal; and 3,4,6-tri-O-Me-Rha bycomparison with authentic samples. 2,3,6-Tri-O-Me-Gal was the main hydrolysis
product of the permethylate and proved that the GalUA units were 1→4 bonded. The absence of dimethyl Gal derivatives
indicated that the MPP′′ structure was unbranched and that Rha was located in the main chain. Based on the results, it was
concluded that MPP′′ was α-(1→4)-polygalacturonan with a (1→2)-bonded Rha unit.
The results from periodate oxidation and molecular weight data for MPP′-1 and MPP′-2 suggested that MPP′-1 had
one branching per three units of the main chain whereas MPP′-2 had four. In general the results indicated that the acidic
components of M. piperita pectin were two branched polymers, the main chain of which was a rhamnopolygalacturonan.
4
The neutral component MPG was chromatographically homogeneous with molecular weight 3.25·10 Da. It did not
contain GalUA. The principal monosaccharides were Gal and Glc in a 1.4:1 ratio. Periodate oxidation of MPG produced
0
.305 mol HCOOH. This corresponded to two branching sites per seven units of the main chain.
Hydrolysis with cation exchanger gave MPG′, the only hydrolysis product of which was Gal. The molecular weight
4
was 1.6·10 Da; degree of polymerization, 98. The absence of hexose in the hydrolysate after CrO oxidation indicated that the
3
Gal units were β-bonded. This was confirmed by an absorption band at 890 cm 1 in the IR spectrum. The MPG′ structure is
linear or slightly branched according to the amount of HCOOH released by periodate oxidation, 0.03 mol per single anhydrous
unit. The presence of erythritol in the hydrolysate from the reduced polyol after Smith degradation indicated that the Gal units
were 1→4 bonded.
–
The main hydrolysis product of MPG′ permethylate was 2,3,6-tri-O-Me-Gal with 2,3,4,6-tetra-O-Me-Gal present in
trace quantities (TLC). This confirmed the conclusion that the main chain of MPG′ was not branched and that this polymer
was the galactan core of MPG.
The bonding of the second dominant monosaccharide Glc to the polygalactan chain was established by studying the
composition of the products from partial hydrolysis of MPG. Ara, Rha, Xyl, Gal, Glc, maltose, maltotriose, and substance G
were detected. Preparative isolation (paper chromatography, PC) and quantitative hydrolysis found that G consisted of Gal and
Glc in a 1:1 ratio. Therefore, Glc was located in side chains. Thus, MPG was a branched β-(1→4)-bonded galactan containing
Glc units in side chains.
Hemicellulose (HC) components were isolated from raw material using basic extraction after removal of PS. This
produced two HC fractions, groups A and B (HC and HC , respectively), the contents of which were 6.98-7.90 (HC ) and
A
B
A
3
.02-3.94% (HC ) of the raw material mass.
B
The principal properties were as follows. HC : carbohydrates, 17.13 ± 0.51%; ash content, 10.86 ± 0.27%; nitrogen,
A
2
4.02 ± 1.12%; [α]D20 -36° (c 0.1, 0.5% KOH); Glc—Xyl—Rha—Ara, 52.3:35.4:7.3:4.9; HC : carbohydrates, 22.04 ± 0.88%;
B
20
ash content, 16.44 ± 0.39%; nitrogen, 13.31 ± 0.89%; [α]D -52° (c 0.1, 0.5% KOH); Xyl—Man—Glc—Gal—Ara,
5
4.3:25.5:14.9:2.8:2.4.
02
5

TABLE 2. Physical Chemical Properties of HC and HCB
A
2
0
Content of MW
Xyl components·10 Da,
[
α]D ,°
Carbohydrate
4
Component CEtOH, % Yield, %
Ara
Gal
Glc Man
Rha
(
c 0.1, 0.5% KOH) content, %
%
of mass fraction
HCA
32.45±4.47
14.95±0.77
10.75±0.57
3.30±0.07
HCA-1
HCA-2
HCA-3
HCA-4
30.0
72.0
80.0
-
8.5
17.5
11.8
6.2
-41
-47
-38
-32
1.4
5.1
7.5
7.7
-
-
-
-
50.5
67.4
50.3
63.4
-
-
-
-
2.1
7.5
9.5
45.9 5.9 (100)
19.9 5.9 (8), 3.1 (92)
32.6 3.1 (17), 1.9 (83)
13.5 1.25 (100)
15.3
HCB
HCB-1
HCB-2
33.0
40.0
22.0
38.1
-67
-58
33.28±0.41
36.89±0.82
8.1
-
-
4.7
14.4 44.1
15.2 14.8
-
-
33.4
65.3
7.0 (100)
4.6 9100)
TABLE 3. Membrane-stabilizing and Antiatherogenic Activity of PS Components
IC , mg/mL
Osmotic resistance of erythrocyte membrane
5
0
Binding of
atherogenic lipids, %
Component
Peroxide hemolysis
Osmotic hemolysis
CNaCl, %
% of control
PS
MPP
0.0546
0.0368
0.0290
0.0752
0.0000
-
0.1691
0.1607
0.1452
2.0724
0.0000
-
0.32
0.29
0.27
0.39
0.41
0.33
0.41
-
+22
+29
+34
+5
0
+20
-
-
24.33±0.11
26.80±0.21
50.22±0.41
6.53±0.24
5.31±0.09
-
MPP′′
MPG
GalUA
Quercetin
Control
Heparin
-
-
-
-
-
100
It is well known that HC and lignin must be present together [5]. It was found that the lignin contents were 42 and
9% for HC and HC , respectively. The carbohydrate contents in the isolated lignin samples were less than 0.5%.
3
A
B
Fractionation of HC using sequential precipitation by ethanol produced four components (HCA-1, HCA-2, HCA-3,
A
and HCA-4) that were colored and insoluble in water (Table 2). Gel chromatography indicated that HC was heterogeneous.
A
The molecular weights of the components fell in a broad range. The negative rotation of [α] 20 and a strong absorption band
D
in the IR spectrum at 889 cm – indicated that β-bonds were present.
1
Fractionation of HCB using gradient percipitation by ethanol isolated two homogeneous (gel chromatography)
components HCB-1 and HCB-2 (Table 2). The principal monosaccharides of both components were Glc, Man, and Xyl in 1:3:2
and 1:1:4 ratios. The negative specific rotation suggested that β-bonds were present.
Themembrane-stabilizing, antiatherogenic, and antioxidant activities ofthe total PS complex, MPP, MPP′′, MPG, and
GalUA were determined experimentally.
PS, MPP, and MPG had positive effects on membrane strength in the peroxide hemolysis model (Table 3). The effect
of PS on osmotic hemolysis was due to the presence of acidic fractions. The MPG activity was over 12 times less than that of
MPP. The last two had a stabilizing effect in the determination of the osmotic resistance of erythrocyte membranes after
incubation with polysaccharide solutions. Acidic components increased the osmotic limit to 0.27% NaCl, which is higher than
the limiting physiological osmotic loading oferythrocytesof0.32-0.34%. GalUAdid not seem capableofstabilizing membranes
up to 0.15 mg/mL. Higher concentrations had a hemolytic effect.
Judging from the antiatherogenic activity of the prepared compounds, PS and its acidic components are more active
than MPG, reaching 24-50% of heparin activity (Table 3).
503

TABLE 4. Antioxidant Activity of PS Components
AOX, %, component concentration, µg/mL
2+
Component
Carotene-H O -DMSO
Fe -ascorbate-SMO
2
2
300
150
30
3
400
200
100
20
PS
MPP
MPP′′
MPG
GalUA
Quercetin
84.84
46.21
2.03
0.00
14.97
100
62.66
36.78
0.00
0.00
13.86
100
23.85
9.24
0.00
0.00
13.31
71.35
0.00
0.00
0.00
0.00
0.00
30.68
39.41
21.76
5.88
17.65
8.82
23.53
19.41
1.18
8.82
2.94
100
17.65
8.82
0.00
4.12
0.00
100
0.00
0.00
0.00
0.00
0.00
54.71
100
Total PS complex had the highest antioxidant activity for β-carotene destruction in DMSO—H O . Its activity was
2
2
2
+
3
3% that of quercetin at a concentration of 30 µg/mL (Table 4). The situation was analogous for Fe -ascorbate-induced
oxidation of sorbitan mooleate. The decrease of AOX for the components was probably due to a decrease in the overall degree
of branching in the complex, which in turn affected that ability to trap free radicals.
EXPERIMENTAL
The aerial part of M. x piperita L. was obtained through a drugstore chain (Travy Bashkirii, batch No. 070604). The
variety was Krasnodar 2.
Isolation of polysaccharides was carried out after removing lipophilic (CHCl :EtOH, 9:1) and alcohol- (80% ethanol)
3
and water-soluble components using a mixture of oxalic-acid and ammonium-oxalate solutions (0.5%, 1:1) with subsequent
concentration of the extracts and precipitation by ethanol (PS). HC were isolated at room temperature using extraction with
KOH (5%). Fraction HC was obtained after neutralization of the extract with HCl (10%); HC , after concentration and
A
B
precipitation by ethanol.
The contents of carbohydrate components were determined by the anthrone method [6]; of phenols, by the literature
method [7] calculated as gallic acid; of protein, by the ninhydrin method after hydrolysis and calculated as alanine [8]; ash
content, bygravimetryafter ashing; acetyl content, byreaction with hydroxylamine [9]; optical rotation, on a Coers polarimeter
with l = 10 cm at 20°C; and pH, using a Checker™1 pH-meter (Hanna Instruments). Spectrophotometric studies were
performed on a Cecil CE 2011 spectrophotometer in 10-mm quartz cuvettes. IR spectra were recorded on a Vector 22
spectrometer in KBr disks (6 mg compound per 600 mg KBr).
PC was carried out on Filtrak FN-2 (carbohydrates) and FN-16(organicacids)paper; TLC, on Silufol (Kavalier) plates;
HPTLC, on Armsorb (Reakhrom) plates impregnated with phosphate buffer (0.07 M Na HPO and 0.07 M KH PO , 96.7:3.3,
2
4
2
4
pH 8.0).
Solvent systems were isopropanol:water (80:20, 1), n-propanol:CHCl :DMSO:phosphate buffer (51:29:11:9, 2),
3
CHCl :CH OH (9:1, 3), CH CO C H :HCOOH:H O (3:1:1, 4), CH CO C H :CH CO H:HCOOH:H O (18:3:1:4, 5).
3
3
3
2
2
5
2
3
2
2
5
3
2
2
Developers were p-hydroxydiphenyl:phosphate (1), diphenylamine:phosphate (2), resorcinol:p-hydroxydiphenyl:
aniline:phthalate:phosphate (3), bromphenol blue (4), KMnO :NaIO :benzidine (5).
4
4
Total hydrolysis of PS and MPP components was carried out as before [10] using compound (100 mg) dissolved in
H SO (10 mL, 80%) for 17 h at 4-5°C, treated with water (70 mL), heated on a boiling-water bath for 4 h, neutralized with
2
4
CaCO , and centrifuged. The aqueous phase was concentrated tothe minimal volume in vacuoat 40°C and analyzed byHPTLC
3
(system 2, developer 3).
The quantitative monosaccharide composition was determined by HPTLC and densitometry using a Mustek 2000
planchette scanner and the TLC-Manager 3.1.1 scanning densitometer program (@PinSoft 2005). Glucose (Roquette),
arabinose, galactose, mannose (Acros Organics) galacturonic acid (Fluka BioChemica), rhamnose (Dia M), and xylose
(Reakhim) were used as standards.
Properties of PS were determined as before [11].
5
04

Gel Chromatography. The studied compound (10 mg) was dissolved in NaCl solution (1 mL, 0.3%) and transferred
to a Sephadex G-100 column (Pharmacia, Uppsala, 1.5 × 60 cm) with elution by NaCl solution (0.3%) at 0.2 mL/min flow rate
and 0.5 mL eluate volumes. The column was calibrated beforehand using Dextran 100000, 70000, 50000, 20000, and 10000
(BioChemica for GPC, Fluka). The internal volume of the column was determined using "blue" dextran (2000000 Da,
Pharmacia, Uppsala). Polysaccharide yields were determined using anthrone sulfate at 625 nm.
Separation of PS Using the Ca Salt. PS (10 g) were dissolved in water (400 mL). The pH was adjusted to 7.5-7.8
using NH solution (20%). CaCl solution (50 mL, 2.5%) was added. The resulting precipitate was centrifuged, washed with
3
2
water, treated with (COOH) solution (400 mL, 2%), and heated at 80°C for 30 min. The precipitate of calcium oxalate was
2
filtered off. The solution was concentrated and precipitated by ethanol (MPP). Yield 3.91 g (39%), [α] 20 +228° (c 1.00,
D
0
(
.1 M NaOH), ash content <0.2%. The solution was concentrated after precipitation of the Ca salt and precipitated by ethanol
MPG). Yield 1.31 g (13%).
Saponification of MPP. MPP (2 g) was dissolved in water (150 mL), treated with NaOH solution (4 mL, 8 M), stirred
continuously at 18-20°C for 30 min, and treated with conc. HCl (4 mL). The resulting precipitate (MPP′) was centrifuged for
1
0 min. Yield 1.84 g (92%), [α]D20 +232° (c 1.00, 0.1 M NaOH).
Isolation of MPP′′. MPP′ (1.5 g) was dissolved in water (100 mL), treated with H SO (70 mL, 3 M), and heated at
2
4
1
00°C for 4 h. The resulting precipitate (MPP′′) was centrifuged and washed with H SO (1%) and then acetone until the
2
4
2
0
rinsings were neutral. Yield 1.02 g (68%), [α]D +239° (c 1.00, 0.1 M NaOH).
Enzymatic hydrolysis of MPP′′ was carried out using α-pectinase (5000 PSU/g, Fluka) bythe literature method [12].
Hydrolysis products were analyzed by PC (system 5, developer 1).
2
0
Isolation of GalUA after enzymatic hydrolysis ofMPP′′ was performed as before [12], mp 153°C (dec.), [α]
+55.4°
D
20
(
c 1.00, H O) {lit. [12] mp 155-159°C (dec.), [α]
2 D
+55.4-55.6° (c 1.00, H O)}. Oxidation by conc. HNO [13] produced a
2
3
compound with mp 222°C, mucic acid.
Acetylation of MPP′′. MPP′′ (100 mg) was dissolved in pyridine (10 mL), treated with acetic anhydride (30 mL), left
at 40°C for 48 h, and treated with HCl (50 mL, 3%). The resulting precipitate was centrifuged, washed with ethanol, and
dissolved in acetone (10 mL). The insoluble part was discarded. The solution was poured into dry diethylether (50 mL). The
acetate precipitate was washed with ethanol and dried, [α] 20 +245° (c 1.0, acetone), K 31.56%.
D
E
Methoxylation, periodate—nitric-acid oxidation of MPP′′, and hydrolysis of MPP′′ acetate were carried out as before
14, 15] to produce samples of methoxylated polygalacturonan {[α] 20 +202° (c 0.5, H O), K 7.15%} and polyaldehyde
[
D
2
M
{
[α]D20 -91° (c 3.0, H O)}.
2
Products of polyaldehyde oxidation were analyzed by PC (system 4, developer 4). Tartaric acid was isolated by
preparative PC [16].
Tartaric acid, mp 170.8-171.0°C, anilide mp 181.0-182.5°C, [α]D20 +12° (c 1.5, H O).
2
Periodate oxidation and Smith degradation were carried out as before [17]. Periodate consumption was calculated
from the absorption decrease at 223 nm [18]; quantitative determination of HCOOH, by titration with NaOH solution (0.01 M).
The hydrolysate after NaBH reduction was analyzed by HPTLC (system 2, developer 5). Oxidation by CrO was performed
4
3
by the literature method [19].
Methylation and Reduction of MPP′′. A suspension of MPP′′ (0.7 g) in methanol (10 mL) was esterified by
diazomethane at +2°C for 24 h. The esterified product was dissolved in water (50 mL) and reduced with NaBH (1 g). After
4
+
1
0 h the solution was passed over KU-2-8 cation exchanger (H -form). The effluent was concentrated in vacuo in the presence
of methanol. The esterification and reduction were carried out 10 times to afford reduced MPP′′ (0.2 g) containing GalUA
10.4%). Reduced MPP′′ and MPG′ were methylated by the Hakomori method [20] and demethylated by the Ciucanu-Kerek
(
method [21]. Reduced MPP′′ (0.2 g) afforded the permethylate (52 mg).
Formolysis and Hydrolysis. The permethylate (50 mg) was heated at 80°C in formic acid (5 mL, 90%) for 1 h.
Formic acid was removed in vacuo in the presence of methanol. The solid was treated with H SO (3 mL, 1 M) and heated at
2
4
1
00°C for 24 h. The hydrolysate was investigated by TLC (system 3, developer 2).
Hydrolysis of MPG by Cation Exchanger. MPG (1 g) was dissolved in water (50 mL) and treated with KU-2-8
H -form) to the level of the solution. The mixture was heated on a boiling-water bath for 2 h, cooled, and filtered. The cation
+
(
exchanger was washed. The solution was concentrated in vacuoand precipitated byacetone. The resulting precipitate was dried
with acetone (MPG′, 450 mg).
505

Partial Hydrolysis of MPG. MPG (100 mg) was dissolved in H SO (15 mL, 0.25 M), heated at 100°C for 5 min,
2
4
and neutralized with CaCO and KU-2-8 cation exchanger. The solution was concentrated in vacuoand analyzed byPC (system
3
1
, developer 1).
Preparative PC was performed on Filtrak FN-8 paper using system 1.
Fractionation of HC by Ethanol. Fraction HC (or HC ) (1 g) was dissolved in KOH solution (100 mL, 5%) and
A
B
gradually treated with ethanol (5 mL each, 95%) until the concentration was 80%. The resulting precipitates were centrifuged
and dried by changing solvent. The solution was acidified byH SO (5%). The resulting precipitate was freed of mineral salts
2
4
+
by reprecipitation from water and treatment with KU-2-8 (H -form).
The lignin content in HC was determined by the Komarov method [22].
Peroxide Hemolysis. Suspensions (1 mL) of rinsed ram erythrocytes in physiological solution (1:150) were treated
-3
with a solution (1 mL) of the compound (0.001-10 mg/mL), H O solution (1 mL, 0.8%), FeSO solution (1 mL, 7.4·10 %);
2
2
4
stirred; and left in the dark at room temperature for 24 h. All solutions were prepared using NaCl (0.9%). Optical density of
the studied solution was determined at 420 nm. Water was used as the reference solution. Optical density of control solutions
was determined in parallel. These were 100% hemolysis, the same composition without adding the component; 0% hemolysis,
erythrocyte suspensions (1 mL); and NaCl (3 mL, 0.9%).
Osmotic Hemolysis. Suspensions (2 mL) of rinsed ram erythrocytes in physiological solution (1:200) were treated
with an aqueous solution (1 mL) of compound (0.001-10 mg/mL) and water (1 mL), stirred, and left in the dark at room
temperature for 24 h. Optical density of the studied solution was determined at 420 nm. Water was used as the reference
solution. Optical densityof controls was determined in parallel. These were 100% hemolysis, erythrocyte suspensions (2 mL);
purified water (2 mL); 0% hemolysis, erythrocyte suspensions (2 mL); and NaCl (2 mL, 0.9%).
Membrane-stabilizingactivitywasestimatedfromtheIC50 (mg/mL), which istheconcentration causing50%inhibition
ofhemolysis. The expressed activitywas estimated using caffeic acid (MRTU6-09-5985-69, Olainsk Chemical Reagent Plant).
Antiatherogenic Activity. Blood serum (0.2 mL) from a healthydonor wasplacedin a10-mL tube, treated with CaCl2
solution (2 mL, 0.025 M), and stirred. Optical density was determined at 410 nm. The solution was treated with the studied
compound (40 µL, 1%) and stirred. Optical density was determined under the same conditions. Heparin (Heparin-Ferein,
Ferein, ZAO Bryntsalov-A, 5000 units/mL) was used as the reference compound.
Osmotic Resistance of Erythrocyte Membranes. A suspension (5 mL) of rinsed erythrocytes from a healthy donor
in NaCl solution (0.9%, 1:100) was treated with a solution of compound (0.5 mL; polysaccharides, 5-10 mg/mL water;
flavonoids, 0.2-0.6 mg/mL in DMSO) and incubated at 37°C for 2 h. Erythrocytes were rinsed with NaCl (0.9%) until the
rinsings were negative for carbohydrates (anthrone sulfate reagent) and flavonoids (Folin reagent). The precipitate of
erythrocytes were placed in a flask with NaCl solution (10 mL, 0.9%) for titration and quickly titrated with water until
completelyhemolyzed (formation ofblood lac). The NaCl concentration in the solution causinghemolysis was determined from
the volume of added water. The control was a sample with added NaCl (0.5 mL, 0.9%).
Antioxidant Activity. A solution (10 mL) ofβ-carotene (water/DMSO, c = 0.012 mg/mL) was placed in a 20-mL tube;
treated with H O solution (5 mL, 0.15%), DMSO (1 mL), a solution of the compound (0.1-1.0), and water to give 20 mL;
2
2
stirred; and stoppered. The mixture was thermostatted at 50°C for 2 h. Every20 min starting at time = 0, samples (2 mL) were
taken. Optical density was determined at 470 nm. The control sample did not contain the compound. Water was used as the
reference solution. The antioxidant activity (%) was calculated using the formula
AOX = [1 - (D 0 - DK120)/(D 0 - DS120)],
are optical densities of the control and studied solutions after 0 and 120 min of incubation.
K
S
where DK0, D
120
, D , and D
S
0
120
K
S
2
+
Fe -ascorbate-induced oxidation of sorbitan mooleate (SMO) was carried out as before [23].
Quercetin (Acros organics) was used as the reference compound for determining the membrane-stabilizing and
antioxidant activity.
ACKNOWLEDGMENT
We thank S. V. Agafonova of the Siberian Institute of Plant Physiology and Biochemistry (Irkutsk) for supplying a
sample of 3,4,6-tri-O-methylrhamnose.
5
06

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