Deglycosylation of isoflavonoid glycosides from maackia amurensis cell culture by β-D-glucosidase from littorina sitkana hepatopancrease

Article abstract of DOI:10.1007/s10600-011-9880-x

Maakia amurensis (strain A-18) cell culture synthesizes a significant quantity of isoflavonoids, a large part of which consists of isoflavone glucosides and malonylglucosides. β-D-Hydrolase enzyme complexes from the marine mollusk Littorina sitkana and the marine mycelial fungus P. canescens were used to obtain isoflavones from their conjugated forms. The specificity of β-D-glucanases from L. sitkana for various glycosides was studied. The deglycosylation efficiency depended on the aglycon structure. The deglycosylated fraction of isoflavonoids obtained from M. amurensis cell culture exhibited antitumor activity.

Full text of DOI:10.1007/s10600-011-9880-x

Chemistry of Natural Compounds, Vol. 47, No. 2, May, 2011 [Russian original No. 2, March-April, 2011]
DEGLYCOSYLATION OF ISOFLAVONOID GLYCOSIDES FROM
Maackia amurensis CELL CULTURE BY ꢀ-D-GLUCOSIDASE
FROM Littorina sitkana HEPATOPANCREASE
*
M. I. Kusaikin, A. M. Zakharenko, S. P. Ermakova, M. V. Veselova,
UDC 577.151.45
E. V. Grigoruk, S. A. Fedoreev, and T. N. Zvyagintseva
Maakia amurensis (strain A-18) cell culture synthesizes a significant quantity of isoflavonoids, a large part
of which consists of isoflavone glucosides and malonylglucosides. ꢀ-D-Hydrolase enzyme complexes from
the marine mollusk Littorina sitkana and the marine mycelial fungus P. canescens were used to obtain
isoflavones from their conjugated forms. The specificity of ꢀ-D-glucanases from L. sitkana for various
glycosides was studied. The deglycosylation efficiency depended on the aglycon structure. The deglycosylated
fraction of isoflavonoids obtained from M. amurensis cell culture exhibited antitumor activity.
Keywords: ꢀ-glucosidase, isoflavonoids, Maackia amurensis, Littorina sitkana, antitumor activity.
Isoflavonoids are an important class of secondary metabolites, the principal structure of which is the 3-phenylchromone
moiety. Isoflavonoids are characterized by carbohydrate substituents on C-7 and C-4ꢁ. Substituents in other positions are
encountered much more rarely [1, 2].
Isoflavonoids occur in plants, for example, in soy, mainly as glucosides and their conjugates whereas isoflavonoid
algycons are used in most pharmacological investigations. This is explained by the more pronounced biological activity of the
aglycons than the glycosides for treating estrogen-dependent diseases [2, 3].
The aglycons can be produced by both acidic and enzymatic hydrolysis of the isoflavonoid glycosides. Acid hydrolysis
or sequential alkaline–acidic hydrolysis is used for total analysis of isoflavonoids in plant sources and biological additives [1].
Enzymatic hydrolysis has indisputable advantages over acidic hydrolysis. All enzymatic reactions occur at physiological
temperatures and pH values. Under such conditions modification of the aglycons is practically excluded and the yield of the
main product approaches 100%.
Multi-year studies by researchers at PIBOC, FEB, RAS, on the pharmacological activities of the polyphenol complex
from Maackia amurensis wood resulted in the pharmacopoeial drug Maksar, which is registered as a hepatoprotective agent.
Cell cultures of M. amurensis were grown at the BSI, FEB, RAS, in order to obtain an industrial source of polyphenols. A cell
culture of M. amurensis (strain A-18) that was obtained from seed sprouts synthesized a significant amount of isoflavonoids,
the composition of which differed from polyphenols isolated earlier from M. amurensis plant. Investigations showed that a
large part of the polyphenols in strain A-18 was present as isoflavonoid glucosides and malonylglucosides [4].
Both pure glycosides (Table 1) and a fraction of isoflavonoid glycosides obtained from M. amurensis cell culture
were used as potential substrates in order to find enzymes capable of catalyzing the hydrolysis of isoflavonoid glycosides. The
fraction contained four glucosides and three malonylglucosides of isoflavonoids.
ꢀ-D-Hydrolase enzyme complexes from the marine mollusk Littorina sitkana and the marine mycelial fungus
P. canescens, which contain ꢀ-D-glycosidases and 1,3-ꢀ-D-glucanases, certain properties and the optimal activity conditions
of which were studied before [5], were used to obtain the isoflavonoid aglycons.
Use of the pure compounds as substrates showed that the enzyme complexes from L. sitkana and P. canescens were
capable of cleaving the O-ꢀ-D-glycoside bond in glucosides of various structures. The glucoside hydrolysis efficiency was
practically the same for both complexes and depended slightly on the structures of the aglycon and carbohydrate (Table 1).
Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, 690022, Vladivostok, fax:
(4232) 31 40 50, e-mail: mik@piboc.dvo.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 182–185, March–
April, 2011. Original article submitted October 26, 2010.
©
0009-3130/11/4702-0197 2011 Springer Science+Business Media, Inc.
197

TABLE 1. Hydrolysis of Isoflavonoids by Enzyme Complexes and Preparations. Accumulation (% of Theoretically Possible)
of Isoflavonoid Aglycons from Their Glucosides and Malonylglucosides
Enzyme complex, %
, %
L. sitkana
Compound
enzyme preparation G-I
enzyme preparation G-II
L. sitkana
P. canescens
100
100
99.63
8.02
90.66
38.88
100
100
100
99.47
100
100
52.68
100
100
100
92.38
44.00
100
100
100
100
100
76.78
27.40
100
10.55
100
1
2
3
4
5
6
7
1
100
80
60
40
20
0
3
5
6
0
50
100
150
200
250
Reaction time, min
Fig. 1. Effect of ꢀ-D-glucosidase G-II from L. sitkana on isoflavonoid
glycosides. Accumulation (% of theoretically possible) of daidzein from
daidzin (1), genistein from genistin (3), formononetin from ononin (5)
and from 6ꢁꢁ-O-malonylononin (6).
For example, the degree of hydrolysis of genistin (3) was less than that of daidzin (1). Modification of the carbohydrate by
introducing a malonyl group into the glucose C-6 position decreased noticeably the degree of enzymatic hydrolysis of modified
genistin and ononin. The hydrolysis efficiency of these substrates was reduced significantly if the enzyme complex from
L. sitkana and pure O-glycoside hydrolases G-I and G-II that were isolated from it were used (Table 1).
Enzyme preparations obtained from the marine mollusk L. sitkana, endo-laminarinase G-I (EC 3.2.1.39) and
ꢀ-D-glucosidase G-II (EC 3.2.1.21), which can participate in cleavage of the glycoside bond, were selected for further
experiments. The selection was based on the knowledge of their catalytic and molecular properties in addition to the ability to
obtain these enzymes in a homogeneous state [5]. Both enzymes are capable of catalyzing cleavage of isoflavonoid glycosides
to form the aglycons (Table 1). The efficiencies of both enzymes for hydrolysis of the glycosides as functions of the aglycon
structure were practically identical. The enzymes also catalyzed cleavage of the glycoside bond with C-6 hydroxyl substitution
in glucose by malonate (4, 6, and 7). However, the enzymes were much less active toward these substrates than toward the
unsubstituted compounds (3 and 5, Table 1).
A comparison of the activity of the enzyme complexes obtained from the marine sources on the total isoflavonoid
glycoside fraction showed that both selected sources contained enzymes that catalyzed efficiently cleavage of the isoflavonoid
glycosides to form the aglycons (Table 1).
ꢀ-D-Glucosidase G-II, which is most sensitive to structural features of isoflavonoid glycosides, was selected for a
detailed study of the influence of structural features of the isoflavonoid glycosides on the rate of the enzyme reaction (Table 1).
Pure preparations of daidzin (1), genistin (3), ononin (5), and 6ꢂ-O-malonylononin (6) were used as substrates. The first three
compounds differed in the aglycon structure; the last, the structure of the carbohydrate, which had a malonic acid substituent
on the glucose C-6 atom.
Figure 1 shows that the rates of hydrolysis of the isoflavonoid glucosides by G-II enzyme were different. The
hydrolysis rate was fastest for 1; slower by 1.7 times, for 3. The initial hydrolysis rate for 5 was less than that of 1 by 1.25
times; for 6, almost 10 times.
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Thus, the aglycon structure had a substantial influence on the hydrolysis rate of the isoflavonoid glycosides by
ꢀ-D-glucosidase G-II. An electron-donating substituent in the 5-position of the aglycon reduced the deglycosylation rate by
almost two times. Replacement of the hydroxyl by methoxyl in the 4ꢂ-position had an insignificant influence of the reduction
of the hydrolysis rate in the initial stages. However, the hydrolysis curve for ononin (5) had an unusual shape (Fig. 1). The
hydrolysis probably occurred in several stages. The first stage had the fastest reaction rate. Then, the accumulation of
formononetin (11) stopped. The amount of it in the incubation medium even decreased slightly. Then, it began to increase again.
The following scenarios explaining such enzyme activity can be envisioned. The decreased hydrolysis rate of ononin
(5) is probably due to inhibition of enzyme G-II by the hydrolysis reaction product formononetin (11). The decrease in the
concentration of 11 during the reaction may be due to an acceleration of the reverse reaction rate, the synthesis of the starting
glycoside, in a certain stage.
We checked both these hypotheses experimentally and found 11 did not inhibit (up to a concentration of 1.25 mg/mL)
the hydrolysis of a synthetic substrate, p-nitrophenyl-ꢀ-D-glucopyranoside, by G-II. It was also established that ꢀ-D-glucosidase
G-II was not capable of synthesizing ononin (5) from p-nitrophenyl-ꢀ-D-glucopyranoside, which was used as a glucose and
formononetin (11) donor. Therefore, the question about the reasons for the unusual behavior of ꢀ-D-glucosidase G-II in the
hydrolysis of 5 remains open.
We showed earlier thatAmur Maackia Dry Extract prepared from M. amurensis exhibits antitumor activity. It seemed
interesting to study the antitumor activity of the isoflavonoid fraction obtained from M. amurensis cell culture and the aglycons
obtained from its enzymatic hydrolysis. Glycoside, isoflavonoid, and aglycon fractions obtained from it by the action of
ꢀ-D-glucosidase G-II did not exhibit cytotoxicity at concentrations up to 28 ꢃg/mL. Also, both fractions did not affect the
growth of cells at concentrations up to 28 ꢃg/mL over 72 h.
The ability of the glycosides and the aglycons obtained from them to inhibit the growth of human breast cancer cells
MDA-MB-231 was determined using the soft agar method that is based on the fact that cancer cells that are not fixed in the
agar layer allow cell colonies to grow whereas compounds with potential antitumor activity inhibit the growth of cell colonies.
The experiment was carried out using MDA-MB-231 cells as before [6] with certain modifications. All compounds were
4
dissolved in DMSO. DMSO was used as a control for treating cells. Cells (2.4 ꢄ 10 cells/mL) were treated with the test
compounds at a concentration of 28 ꢃg/mL per milliliter of BME (Basal Medium Eagle) agar (0.33%) over BME agar (0.5%,
3.5 mL) containing extracts of the compounds at a concentration of 28 ꢃg/mL. Cells were cultivated at 37°C and 5% CO for
2
21 d. Cell colonies were estimated using an inverted microscope (Motic AE20, China) and Motic Images Plus software. Two
independent experiments with three samples at each concentration were used for each compound.
Aglycon fractions at a concentration of 28 ꢃg/mL inhibited growth of MDA-MB-231 human breast cancer cell colonies
by 60% compared with the control whereas the glycoside fraction at this concentration did not inhibit the growth of their
colonies. These results indicated that the aglycon fraction had antitumor activity against MDA-MB-231 human breast cancer cells.
EXPERIMENTAL
Analytical Methods. Reducing sugars were determined by the Nelson method [7]; protein content in preparations,
by the Lowry method [8].
Substrates. p-Nitrophenyl-ꢀ-D-glucopyranoside was purchased from Sigma (USA). Pure isoflavonoid glucosides
were prepared by the literature method [4].
Preparation of Isoflavonoid Glucoside Fraction. We used strain A-18 of M. amurensis cell culture. Dried cell
culture was extracted three times with CHCl :EtOH (3:1) over 2 h. The combined extracts were lyophilized and separated
3
preparatively over a column of Toyopearl 50F (3 ꢄ 40 cm) with elution by a linear gradient of H O:EtOH (containing 0.04%
2
HCOOH). The fraction eluted by 20% aqueous EtOH contained only isoflavonoid glucosides. The composition of the
fraction was determined using HPLC.
Enzyme complexes of ꢀ-D-hydrolases from L. sitkana and P. canescens in addition to ꢀ-D-glucosidases from
L. sitkana were obtained by the literature method [5].
Determination of Enzyme Activity. The ꢀ-glucosidase activity was determined from the amount of p-nitrophenol
released by the action of the enzyme on p-nitrophenyl-ꢀ-D-glucopyranoside [5]. The activity unit was taken as the amount of
enzyme that catalyzed the formation of 1 nmol of product (p-nitrophenol) during 1 h under the determination conditions.
Products from Enzymatic Hydrolysis of Isoflavonoid Glucosides. A weighed portion (4 mg) of isoflavonoid
–2
glycosides (4 mg) was dissolved in sodium succinate buffer (0.025 M, pH 5.4), treated with ꢀ-D-glucosidase (2 ꢄ 10 U), and
199

incubated for 18 h at 37°C. The reaction was stopped by adding CH CN. Then the mixture was lyophilized and analyzed by
3
HPLC.
Analytical HPLC was performed on anAgilent Technologies chromatograph (Series 1100) equipped with a QuatPump
G1311A high-pressure pump system, G1322A Degasser, G1328B injector, and G1314A VWD detector. The analysis results
®
were processed using ChemStation software ver. 09 (Germany). We used the literature method [4] and dihydroquercetin
(DHQ) as an internal standard.
–3
Inhibition of ꢀ-D-Glucosidase G-II by Formononetin. Enzyme solution (20 ꢃL, 10 U) in sodium succinate
buffer (0.025 M, pH 5.2) was treated with inhibitor solution (10 ꢃL) containing from 0.5 to 200 ꢃg of compound in sodium
succinate buffer (0.025 M, pH 5.2). The mixture was held at 37°C for 10 min, treated with p-nitrophenyl-ꢀ-D-glucopyranoside
(200 ꢃL, 1 mg/mL) in sodium succinate buffer (pH 5.2), and incubated at 37°C for 30 min. The residual activity was determined.
Enzymatic Synthesis of Glucosides by ꢀ-D-Glucosidase G-II. The appropriate aglycon (1 mg) and p-nitrophenyl-
–5
ꢀ-D-glucopyranoside (5 mg) were dissolved in enzyme solution (1 mL, 5 ꢄ 10 U) and incubated at 37°C for 20, 40, and
120 min and 24 h. Production of the glycosides was monitored by HPLC.
Cell Cultivation. HCT-116 human colon cancer cells (ATCC No. CCL-247) and MDA-MB-231 human breast
cancer cells (ATCC No. HTB-26) were cultivated in McCoy 5a and DMEM (Dulbecco’s modified Eagle’s medium), respectively,
and 10% phosphate buffer (FBS) with added penicillin (100 U/L) and streptomycin (100 ꢃg/L) in an MCO-18AIC incubator
(Sanyo, Japan) at 37°C with 5% CO content.
2
5
Determination of Isoflavonoid Cytotoxicity. HCT-116 and MDA-MB-231 cells (1.5 ꢄ 10 /mL) were inoculated
into 96-well plates and cultivated in 10% McCoy 5a and DMEM (200 ꢃL), respectively, in a CO incubator at 37°C for 24 h.
2
Then, cells were treated with isoflavonoids at concentrations of 3, 5, 7, 14, and 28 ꢃg/mL in fresh medium and incubated for
24 h. Each well was treated after incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS reagent) (15 ꢃL) and incubated for 4 h (37°C, 5% CO ). The optical density was measured on a Bio-Tek
2
Instruments spectrophotometer (USA) at wavelength 490/630 nm (A
).
490/630
Neoplastic Cell Transformation (Soft Agar Method). The effect of the isoflavonoids on the formation and growth
of colonies of MDA-MB-231 human breast cancer cells was determined by the soft agar method [6]. MDA-MB-231 cells
4
(2.4 ꢄ 10 /mL) were treated with isoflavonoids (28 ꢃg/mL) in agar (1 mL, 0.33%) containing BME (1.8%), containing 10%
over agar (3.5 mL, 0.5%) with BME (1.8%), and containing FBS (10%) and isoflavonoid (28 ꢃg/mL). Controls were treated
with the corresponding amount of DMSO. Cells were cultivated at 37°C and 5% CO for 21 d. Human colon cancer cell
2
colonies were estimated using a Motic AE20 inverted microscope (China) and Motic Images Plus software.
Statistical data treatment was carried out using the Student t-criterion with 95% probability limits (SigmaPlot
2000, version 6, SPSS Inc., USA).
ACKNOWLEDGMENT
The work was supported by RFBR grant 09-04-00761, RAS Presidium Program Molecular and Cellular Biology, and
Presidential Grant MK 1472.2009.4.
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