INTRODUCTION

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 [3.2.1.21] 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 β–glycosidic 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 standards.

Table 1:	Digestion of glycosides by plant isoenzymes

RESULTS AND DISCUSSION

The leaves extract of G. arborea was fractionated using a liquid–liquid 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 (3.2.1.161) (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 an α-lα-rhamnosyl-1,6-β-dα-glucoside.

Fig. 1:	Structure and hydrolysis of compound 1 and 2

Fig. 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.

Fig. 3:	(a-d) Hydrolysis of Compound 2 by β-glucosidases, as analyzed by TLC

Table 2:	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 as well.

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.

ACKNOWLEDGMENTS

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.