Structure Confirmation of Rare Conjugate Glycosides from Glycosmis arborea (Roxb.) with the Action of β-Glucosidases
J.R. Kedutat Cairns
Two flavanones, 5,5′-dihydroxy-4′-methoxy-7-O-(α-L-rhamnosyl-(1′′′>6′′)-β-D-glucopyranosyl) flavanone 1 and (5,5′-dihydroxy-4′-methoxy-5-O-β-D-glucopyranosyl) flavanone 2, were isolated by liquid chromatography from ethanolic extracts of Glycosmis arborea (Roxb.) leaves and tested for hydrolysis by various β-glucosidases. The β-glucosidases from Dalbergia cochinchinensis, Dalbergia nigrescens, barley, rice and almond were compared for their ability to hydrolyze the conjugated flavanone glycosides from G. arborea by thin layer chromatography of the reaction products. The disaccharide glycoside, compound 1 was only hydrolyzed by the D. nigrescens β- glucosidase, which appeared to release the sugar mainly as a disaccharide, while compound 2 was hydrolyzed by all β-glucosidases tested, except Os9BGlu31. These compounds also showed significant antifeedant activity toward the polyphagous crop pest Spodoptera litura. This is the first report about the structural elucidation using enzymatic studies of bioactive flavanone glycosides from G. arborea.
to cite this article:
D.S. Negi, A. Kumar, R.K. Sharma, N. Shukla, N. Negi, M.L. Tamta, Y. Bansal, Por-gnam Prasert and J.R. Kedutat Cairns, 2011. Structure Confirmation of Rare Conjugate Glycosides from Glycosmis arborea (Roxb.) with the Action of β-Glucosidases. Research Journal of Phytochemistry, 5: 32-40.
Received: October 30, 2010;
Accepted: December 21, 2010;
Published: February 26, 2011
Glycosmis arborea (Roxb.) is an evergreen shrub belonging to Rutaceae
family and is distributed in warm and temperate regions of India. Roots of this
plant are traditionally used to overcome from facial inflammation, rheumatism,
anaemia and jaundice. Leaves are useful in treatment of fever, eczema, skin
disease, wounds and hepatic disease. Fruits of this plant are used to treat
cough and bronchitis (Warrier et al., 1995). Previously,
carbazole alkaloids and quinoline alkaloids were isolated from the roots of
G. arborea (Chakravarty et al., 1999).
A hepatoprotective carotenoid was also isolated from this plant (Kivotani
et al., 1996).
The tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera:
Noctuidae) is a serious polyphagous pest, infesting more than 150 species and
is widely distributed in India (Kumar et al., 2007).
It causes significant losses of crops from 26-100% (Dhir
et al., 1992), it is also reported to be attacking cotton in North
India (Arora, 1993). Pesticides have played a significant
role in increasing agricultural production. However, the continuous use of insecticides,
has not only caused death through poisoning and their persistence in environment
and food chain, but also caused resistance in pest populations (Wei
et al., 2004; Kranthi et al., 2002).
Given this circumstance, research needs to be aimed at establishing alternative
means of insect control. One promising means is to find phytochemicals from
plants and aqueous extracts of the leaves of the medicinal plants Ipomea
carnea (L.), Pedalium myrex (L.) and Adhatoda vasica (L.)
could have both toxic and antifeedant effects on this pest (Sujatha
et al., 2010). An antifeedant is a phytochemical agent that causes
a pest to stop eating (Isman et al., 1996). Several
plants have been reported to produce a diverse range of secondary metabolites,
such as terpenoids, alkaloids polyacetylenes flavonoids, amino acids and sugars
that protect the plants from infestation by insects (Patel
and Patel, 1996).
β-Glucosidases [188.8.131.52] play significant roles in plants, including
growth regulation, response to stress, lignifications, cell wall β-glucan
degradation, and defense (Cairns and Esen, 2010). These
enzymes act on βglycosidic bonds linking a glucosyl residue to glucose-substituted
molecules such as oligosaccharides and aryl and alkyl glucosides, with different
specificities. Hessler et al. (1997) reported
that β-glucosidase from Saccharopolyspora eryraea could hydrolyze
genistin during fermentation of soy-based media, a β-glucosidase from bifidobacteria
in soy milk was capable of converting glucosides to their aglycones 990 m, 590
and 1378 m, respectively (Tsangalis et al., 2002).
Pandjaitan et al. (2000a, b)
treated soy protein isolate β-glucosides with almond β-glucosidase
to convert most of its isoflavone glucosides to their aglycone. Suzuki
et al. (2006) purified, cloned and characterized β-glucosidase
from soybean that could hydrolyze isoflavone conjugates, with high activity
toward 6=-O-malonyl genistin. Isoflavonoid β-glucosidase have been
described from the seeds of Dalbergia cochinchinensis and Dalbergia
nigrescens (Chuankhayan et al., 2005, 2007a,b).
These enzymes had high hydrolytic activity on isoflavonoid glycosides from the
same seeds and the D. nigrescens enzyme can efficiently hydrolyze β-glycosides
to remove 6-O-modified glucosyl residues. Most other plant β-glucosidases
are not known to be able to hydrolyze these more complex glycosides. In present
communication we evaluated the structure and linkage of isolated compounds from
G. arborea by β-glucosidases with different specificities.
MATERIALS AND METHODS
General experimental procedure: The UV and IR spectra were recorded
on Beckman 64 UV spectrophotometer and Perkin-ElmerRX-1 spectrophotometer. NMR
spectra were measured on Bruker AVANCE 500 MHz spectrometer. JEOL JMS 600 H
was used for MS data. Melting points (uncorrected) were measured on Complab
Melting point apparatus. Silica gel 60-120 (Merck) used for isolation of compounds
by column chromatography. TLC analyses were carried out using aluminum-backed
silica gel 60 F254 (0.20 mm thickness) plates (Merck).
Plant material: Leaves of G. arborea were collected from Rajaji National Park, Rishikesh, Uttarakhand, India (during flowering season at altitude of 2300-2500 m in October 2005) and identified by Prof R. D. Gaur, a taxonomist of Department of Botany, HNB Garhwal University. The voucher specimen (8977, GUH) have been deposited in the herbarium of the Department of Botany, HNB Garhwal University.
Leaf extraction and isolation: The air dried leaves were grinded and
extracted with 90% EtOH. The total ethanolic extract was concentrated under
reduced pressure at a temperature below 50°C to a dark green viscous mass
that was partitioned with hexane and n-butanol. The butanol soluble layer was
then fractionated into chloroform, ethyl acetate and methanol soluble fractions.
Methanol fraction was subjected to column chromatography over 60-120 mesh silica
gel (Merck) and eluted with chloroform and MeOH with the increasing polarity.
Fractions were collected (100 mL each) and those with similar TLC patterns were
mixed together. Compound 1 (46 mg) and 2 (36 mg) were obtained from CHCl3:
MeOH (85:15) eluate.
β-Glucosidase enzymes: Almond β-glucosidase was purchased
from Sigma, while other β-glucosidase enzymes were produced from recombinant
expression systems and seed extracts. Rice β-glucosidases Os3BGlu7, Os4BGlu12
and Os9BGlu31 were produced as thioredoxin fusion proteins in Escherichia
coli and purified as previously described (Opassiri
et al., 2003, 2004 2006).
rHvBII was similarly produced in the same system as previously described (Kuntothom
et al., 2009). DnBGlu was purified from D. nigrescens seeds
(Chuankhayan et al., 2005), whereas the recombinant
enzyme DnBGlu2 was expressed in Pichia pastoris (Chuankhayan
et al., 2007a). Thai rosewood (Dalbergia cochinchinensis)
β-glucosidase (dalcochinase, TRW) (Srisomsap et
al., 1995; Svasti et al., 1999) was expressed
in P. pastoris and purified by hydrophobic interaction chromatography
and IMAC (Toonkool et al., 2006).
Antifeedant activity: The antifeedant activity of the extracts against the polyphagous pest S. litura was tested by the leaf dip method. Five percent concentrations of each extract were prepared by dissolving extracts in a small quantity of ethanol and diluting in water containing 0.05% TritonX100. The leaf discs of about 5 cm2 were prepared out of castor leaf (Ricinus communis L.) and were dipped for 30 sec in an extract or compound separately. The leaf discs dipped only in water containing 0.05% TritonX100 were used as controls. The leaf discs were air dried and on each treated leaf disc ten larvae of S. litura (one day old) were released. Three replications were maintained for each extract. Larval weight was taken after four days of treatment.
Percent growth reduction was calculated in comparison to control as shown below
and the values given in Table 1:
Hydrolysis of compounds: To compare the hydrolytic efficiency of Dalbergia
rice, barley and almond β-glucosidases towards the isolated compounds,
10 μg aliquots of the compounds were separately hydrolyzed with 0.001 unit
of a β-glucosidase in 100 μL of 0.1 M sodium acetate, pH 5.5. The
reaction mixtures were incubated at 37°C for 10 min or 16 h, boiled 5 min
to stop the reactions, then dried by speed vacuum and resuspended in 100 μL
of 10% acetronitrile in 0.1% phosphoric acid/water. A control reaction of water
without enzyme was set up in the same manner. The hydrolyzed products were evaluated
by Thin-Layer Chromatography (TLC) on analytical silica gel 60 F254
aluminum plates (Merck, Darmstadt, Germany) with one of two different solvent
systems, which were (A) ethanol: methanol: acetic acid: water (7.5:0.5:1:1)
and (B) butanol: methanol: acetic acid: water (7.5:0.5:1:1). Flavonoids were
visualized by absorbance under UV light and carbohydrates were visualized by
spraying with 10% H2SO4 in methanol and incubating at
110°C for 20 min sugars were tentatively identified by comparison with commercial
|| Digestion of glycosides by plant isoenzymes
RESULTS AND DISCUSSION
The leaves extract of G. arborea was fractionated using a liquidliquid
partition procedure with solvents of increasing polarity. The methanol fraction
was then purified using open column chromatography over silica gel 60-120 mesh
to yield two flavanone glycosides 1 and 2. Compound 1 has molecular formula
C28H34O15 it was identified as 5,5′-dihydroxy-4′-methoxy-7-O-(α-L-rhamnosyl-(1′′′>6′′)-β-D-glucopyranosyl
flavanone, whereas compound 2 with molecular formula C22H24O11,
was characterized as 7,5′-dihydroxy-4′-methoxy-5-O-β-D-glucopyranosyl flavanone
(Fig. 1.) both are known compounds and their structures were
confirmed on the basis of comparison of their spectroscopic data with reported
literature (Wei et al., 2007; Jovanoic
et al., 1994).
Both compounds are firstly reported from this plant. For the confirmation of
glycoside linkage we digested 1 and 2 by commercial almond β-glucosidase
or rice Os3BGlu7, Os4BGlu12 and Os9BGlu31 or barley rHvBII β-glycosides
on incubation at overnight 30°C the compound 1 was not digested by these
enzymes, whereas 2 was digested by Os3BGlu7, Os4BGlu12 and rHvBII, to further
confirm the glycoside linkage, we digested the compounds with DnBGlu, DnBGlu2
and TRW (Thai rosewood). TLC showed that the recombinant enzyme DnBGlu2 could
completely digest compound 1 (spot three, Fig. 2a), while
DnBGlu from seed digested it slightly (Fig. 2b, spot nine).
DnBGlu is an isoflavone 7-O-β-D-glucosidase and acuminosidase (184.108.40.206)
(Chuankhayan et al., 2005).
Recombinant DnBGlu2 and DnBGlu can cut glucose, 6-acetyl-glucose, 6-malonyl-glucose
and 6- β-dα-apiosyl-β-dα-glucose off from the 7 hydroxyl
of isoflavonoids (Chuankhayan et al., 2005, 2007a).
The digestion of compound 1 by DnBGlu2 suggested that compound may contain a
6-O-modified glucose for the sugar, since DnBGlu2 hydrolyzes flavonoid 6-β-D-apiosyl-β-D
glucosides and 6-malonyl glucosides much better than Thai rosewood (Dalbergia
cochinchinensis) β-glucosidase (TRW, dalcochinase) (Srisomsap
et al., 1995) and the other enzymes could not hydrolyze these types
of sugars. β-Glucosidases from almond, D. nigrescens (DnBglu and
DnBglu2) and D. cochinchinensis (TRW) were also able to hydrolyze compound
2. Digestion of both compounds is shown in Table 1. This inferred
that enzyme DnBGlu2 efficiently hydrolyzed the α-lα-rhamnosyl-1,6-β-dα-glucosyl
moiety from the 7-O of the flavanone. To our knowledge, this is the first time
that DnBGlu, DnBGlu2 or any other β-glucosidase has been reported to hydrolyze
|| Structure and hydrolysis of compound 1 and 2
||The hydrolysis of Compound 1 by Dalbergia β-glucosidases.
Solvent system A [ethanol methanol: acetic acid: water (7.5:0.5:1:1)] was
used to separate the glycosides and aglycones in TLC 1a, while solvent system
B [butanol: methanol: acetic acid: water (7.5:0.5:1:1)] was used in TLC
1b. Compound 1 was digested overnight with DnBGlu (1a spot 2 and 1b spot
8), recombinant DnBGlu2 (1a spot 3 and 1b spot 9), Thai rosewood β-glucosidase
(1a spot 4 and 1b spot 10), and almond β-glucosidase (1a spot 5 and
1b spot 11). The control digest (Compound 1) is shown in Fig.
3a spot 1 and 3b spot 7, while sugar standards are
shown in Fig. 2b as follows: 1, glucose; 2, cellobiose;
3, lactose; 4, galactose; 5, sucrose; and 6, maltose
It is not completely unexpected, since the enzymes natural substrates appear
to include α-lα-apiosyl-1,6-β-D-glucosides (Chuankhayan
et al., 2005, 2007b), but it has not been
previously demonstrated. This suggests that the active site can accommodate
the binding of a range of sugars, in addition to malonyl and acetyl groups bound
to the 6-O of the glucosyl residue. This makes it useful for determining whether
a given flavonoid 7-O-glycoside has a (1→6)-linked disaccharide or glucosyl
group, by comparing the hydrolysis by DnBGlu2 to that by one of the many β-glucosidases
that can only hydrolyze flavonoid glucosides.
|| (a-d) Hydrolysis of Compound 2 by β-glucosidases, as
analyzed by TLC
|| Antifeedant activity of leaf extracts of G. arborea and
its compounds against S. litura
|Experiment done in triplicate, ±SD value
Os3BGlu7 is best at hydrolyzing long celloligosaccharides, but can hydrolyze
some glycosides such as pyridoxine glycoside (Opassiri et
al., 2004). It has β-D-glucosidase, β-D-fucosidase, β-D-mannosidase
and β-D-galactosidase activities. Os4BGlu12, another rice isoenzyme expressed
in germinating shoots, also hydrolyzes oligosaccharides and has β-fucosidase,
β-glucosidase, β-galactosidase, β-xylosidase and α-L-arabinosidase
activities (Opassiri et al., 2006). Barley rHvBII
is a recombinant enzyme nearly the same as barley BGQ60 (Leah
et al., 1995) and barley β-glucosidase isoenzyme II (Hrmova
et al., 1996, 1998). It is a β-D-mannosidase
with β-D-glucosidase, β-D-fucosidase and β-D-galactosidase activities
In Fig. 3a and b, the TLC was run in solvent
system A, while 3c and d show the TLC run in solvent system B. 3a and c are
the images of the TLC taken under UV light, which show the dark absorbing spots
for the glycosides and the bright fluorescent spots for the aglycone, while
3b and d show the TLCs after development with 10% sulfuric acid and heating.
Sugar standards are shown in TLC 3c and d as follows: 1, glucose; 2, apiose;
3, cellobiose; 4, lactose; 5, galactose; 6, sucrose and 7, maltose. The control
reaction and unreacted substrate are shown in Lanes 1 and 7 in 3a and b and
the control reaction in lane 8 (Compound 2) in Fig. 3c and
d. The enzymes used to digest were Os4BGlu12 (Fig.
3a and b lanes 2 and 5, Fig. 3c and
d lanes 9 and 12); Os3BGlu7 (Fig. 3a and
3b lane 3 and C and D lane 10), barley rHvBII (A and B lane
4 and C and D lane 11) and Os9BGlu31 (A and B lane 6 and Fig.
3c and d lane 13).
An evaluation of the antifeedant activities of extract, fractions and isolated
compounds from G. arborea on S. litura larvae was also carried
out and the results are shown in Table 2. The chloroform extract
had significant activity and showed the maximum mortality of 50% and a growth
reduction of 93% for G. arborea. The hexane and butanol extracts caused
lower mortalities and reductions in growth rates, while in the pure compounds
did not cause mortality but cause growth reduction in the larva. It should be
noted that compounds in water extracts from other Indian medicinal plants have
also been shown to have toxic and antifeedant activities toward S. litura
larvae (Sujatha et al., 2010). However, the addition
of more antifeedant compounds to combat this pest should be useful.
Authors are thankful to Director, Uttarakhand Council for Science and Technology (UCOST), Uttarakhand, India and University Grants Commission (UGC) New Delhi for financial assistance. Phimonphan Chuankhayan, Pornphimol Metheenukul, Teerachai Kuntothom, Sukanya Luang, Salila Pengthaisong, Thipwarin Rimlumduan, and Sompong Sansenya are thanked for production of enzymes. Support for enzyme work that was provided by the Thailand Research Fund and Suranaree University of Technology, Thailand.
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