TOXIC EFFECTS OF THREE SELECTED MALAYSIAN TIMBERS PLANT EXTRACTS AGAINST SUBTERRANEAN TERMITES

The toxic effects of selected Malaysian timbers (Madhuca utilis, Anisoptera laevis and Endospermum malaccense) heartwood extracts were studied with the aim to determine and understanding the function of wood extracts as a natural protection against termite. The results show that no-choice experiments revealed toxic properties of all investigated extracts by the contact against Coptotermes gestroi and Coptotermes curvignathus. However, high termite mortality was only achieved with Madhuca utilis extracts and methanol solvents.


INTRODUCTION
Extractives are low molecular weight compounds that can be extracted by polar or non-polar solvents and encompass complex, and had diverse physical properties (Fengel and Wegener 1989).Because of this, many different solvents and mixtures have been used for extraction.Solvents such as hexane and acetone/water mixtures have been used with some tropical timbers (Kilic and Niemz 2010); methanol and hexane with Azadirachta excelsa (Ahmad Said et al. 2006) and ethanol-benzene with Shorea ovalis and Neobalanocarpus heimii (Ahmad-Said and Mohd-Hamami 1982).In addition, structure, distribution and quantity of secondary metabolites are useful markers for chemotaxonomy (Banthorpe et al. 1972).
It is known that wood extracts are a major contributory factor in the natural durability of the wood (Scheffer andCowling 1966, Hillis 1987).Many studies (Carter et al. 1975, Steller and Labosky 1982, Chang et al. 2001, Chang and Cheng 2002, Watanabe et al. 2005, Elango et al. 2012, Roszaini et al. 2014, Roszaini et al. 2015) show some promising result on wood extractive against termites.They found that bark and Heartwood extractives exhibited antitermitic activity in a certain percentage of concentrations.Meanwhile, Arango et al. (1992) reported that several advantages can be obtained from the application of wood extractives as wood preservatives.It is relatively safer than synthetic preservative, but still effective.It is Barnes (1992) also easier to detoxify and dispose off without adverse environmental effects because it's the organic based materials.
In some instances, it is only present in small amounts (3 to 6% of oven-dry weight) (Rudman 1965, Mori et al. 1997, Reyes-Chilpa et al. 1998, Celimene et al. 1999, Windeisen et al. 2002, Haupt et al. 2003, Neya et al. 2004, Mburu et al. 2007).In other cases, Roszaini (2011) found in her study that this value can achieve up to 15% (of oven-dry weight) for heartwood and to 35% (of oven-dry weight) for bark of tropical timbers.Phenolic compounds, terpenes, carbohydrates, long-chain fatty acids, waxes and other substances, including steryl esters and sterols are among the mainly chemical compounds in heartwood extractives (Fengel and Wegener 1989).Many single or groups of extraneous compounds are known to inhibit the activities of biological agents.For example, sesquiterpenes possess a wide spectrum of biological activity, playing a role in plant defense mechanisms against insects and fungi (Fraga 2003, Wu et al. 2005) and, pinosylvin and pinosylvin-monomethyl-ether have been found toxic to fungi (the Sirex fungus, presumably Amylostereum sp.) (Hillis and Inoue 1968).
Meanwhile, Madhuca utilis (Ridley) H.J. Lam ex K. Heyne known as bitis (local name) is one of the large trees which can achieve up to 50 m in height, 1 m in diameter and 2 m high of buttresses (Orwa et al. 2009).It belongs to the family of Sapotaceae and classified as heavy hardwood with a density of 820 -1,120 kg/m 3 air dry (Lim et al. 1998).
Anisoptera laevis (Ridl) which is also known as merawan under Malaysian common name is an evergreen tree with a relatively small crown.The tree belongs to the family of Dipterocarpaceae and locally distributed in lowland primary forest of Peninsular Malaysia (Ken 2014).The timber is a medium hardwood with a density of 495-980 kg/m 3 air dry.Its wood is hard and heavy, and particularly used for bridge, rafters, joists, door and window frames, flooring, joinery, furniture manufacture, veneer and plywood manufacture (Wood identification 2010).
Endospermum malaccense Miq.(sesendok) is a timber belonging to the family of Euphorbiaceae.It can be found in lowland to low-Montane forest (up to 1000m altitude) and in all states of Peninsular Malaysia (except Perlis) (Mohd Shukari 1982).It's also one of the timber species that has been proposed for plantation in Peninsular Malaysia as an alternative timber species to rubberwood with an excellent working and nailing properties (Ahmad Zuhaidi et al. 2002, Khairul et al. 2010).
However, to the best of our knowledge, a study of the wood extracts of M. utilis, A. laevis and E. malaccense extracted with different solvent from Malaysia, or any other country, has not been reported to date.One study done by Roszaini et al. (2014) shows some promising as antitermitic of M. utilis when extract with toluene/EtOH but, they do not include other solvents in their study.In another study, methanol extraction of E. malaccense showed highest antifungal activity against a white-rot fungus, Pycnoporus sanguineus, at a minimum effective amount of 100 μg (Kawamura et al. 2011).No single study was done for E. malaccense and A. laevis wood extracts against termite.On the other hand, studies (Naczk and Shahidi 2004, Spigno et al. 2007, Kajdžanoska et al. 2011, Lolita et al. 2012) have shown that different solvent extracts different compounds.Based on their studies, it is very important to find the best solvents for extraction of these compounds from plants especially for tropical timbers.
The objective of this paper is to investigate the effects of different solvent extractions on some wood species with respect on the feeding behavior of the two Asian subterranean termites Coptotermes gestroi and C. curvignathus.Although M. utilis, A. laevis and E. malaccense have been reported in durable, moderate and non-durable class timbers respectively, its antitermitic activity has not been tested.So this would be the first study of the antitermitic activity of crude extract of different Malaysian wood species against the two aggressive Asian subterranean termites.

Termite
Two subterranean termites, Coptotermes gestroi Wasmann and C. curvignathus Holmgren (Isoptera: Rhinotermitidae), were collected from active field colonies at the Forest Research Institute Malaysia (FRIM) campus using a method described before (Roszaini et al. 2009).

Wood block bioassay against subterranean termites
The un-extracted or had previously been extracted (extracted for 8 hours in an orbital shaker) wood blocks (25 mm x 25 mm x 6 mm) were subjected to no choice feeding tests according to ASTM D3345-08 (ASTM 1988) standard methods with slightly modified.Rubberwood (Hevea brasiliensis) were used as controls.Screw-top bottles of 8 cm in diameter by 13 cm high were filled with 200 g of sterilized sand and 30 ml distilled water.The bottles were left overnight to equilibrate to laboratory conditions before test initiation.One block of each timber species were placed on the surface of the damp sand and 400 termites (360 workers and 40 soldiers) were added to each bottle.All bottles were stored in an incubator maintained at 22+2 o C and 65+5% relative humidity for 28 days.Within this period, if it was found that all termites appeared dead, the bottle would be taken out and the number of days until 100% mortality would be recorded.At the end of the fourth week the blocks were removed, cleaned, dried overnight and reweighed.The remaining live termites were weighed and recorded for each of the bottles.Then the wood blocks were classified according to the standard method used (Table 1).

Extraction and isolation
All the heartwood timber species were ground to fine sawdust powder, passed through a 250 mesh sieve and dried at 60 o C (to avoid the possibility of extracts degradation) before extraction.About 50 g of wood sawdust was extracted with four different solvents [absolute methanol (MeOH), absolute ethanol (EtOH), acetone and petroleum ether (PETETHR)] for 8 hours used an orbital shaker (Gallenkamp, UK).The extracts were concentrated under reduced pressure at 45 o C, using a rotary evaporator (EYELA, SB-651, Rikakikai Co. Ltd.Tokyo, Japan) and stored in a refrigerator (-4 o C), until used for analyses.Weight losses of samples were calculated from the oven dry weights at 60 o C (48 hours) before and after the extraction.Retention of extractive material was calculated (mg/m 3 ) as follows: R = (M 1 -M 0 ) x C (1) V where, M 1 is weight after treatment (g), M 0 is a weight before treatment (g), C is concentration levels of solutions and V is volume of filter paper (m 3 ).The extractive retentions (mg/m 3 ) in treating filter paper as calculated by solution uptake are presented in Table 2.  0,5  0,38 (0,25)  1,78 (0,26)  0,95 (0,25)  1

Antitermitic bioassay (Toxicity determination)
The bioassay method used by previous studies (Roszaini et al. 2013) with slightly modified was used to evaluate the antitermitic activity of wood extracts against C. gestroi and C. curvignathus.Samples of 5,0 mg, 10 mg, 20 mg, 30 mg and 40 mg of wood extract from four different wood species were dissolved in 100 μl of MeOH to obtain solutions (m/v) of 0,5%; 1,0%; 2,0%; 3% and 4%, respectively.Then 20 μl of the solutions were applied to each 30 mg filter paper samples (Advantec, 8 mm diameter and 1,5 mm thickness) and dried in vacuum desiccators for 24 hours.The paper discs were weighed before and after drying.Untreated paper discs were used as a control.20 active termite workers were introduced into each Petri dish (90 mm diameter and 16 mm height) which contained 3 g of sterile sand.A few drops of water were added periodically to the basal edge of each Petri dish.All the Petri dishes with covers were placed into an incubator (maintained in darkness) at 22+2 o C and 65+5% RH and the mortality of the termites was counted and recorded every 24 hours for 10 days.Each test contained 5 replicates including the control.The consumption of the filter papers was calculated from the difference in dry weights before and after the exposure.A dose-mortality line was developed depends on the exposure time(s) and the lethal concentration (LC 50 ) of wood extracts was determined using the probit method (Finney 1971).

Statistical analysis
One way analysis of variance (ANOVA) was performed on all data to determine the significance of variation in extracting compounds and antitermitic between wood species as well as between samples using MINITAB 15 computer programme.The LC 50 values were determined directly from probit Maderas.Ciencia y tecnología 19(4): 417 -432, 2017 Toxic effects of three selected..: Kadir analysis or calculated by substituting 50% for "y" into the curve equation in the graph.

Extractives yield
The quantities of extractive yield of three tropical timber species are presented in Table 3. Table 3 shows that MeOH yielded greater amounts of extractive than the other three solvents (EtOH, PETETHR and acetone) in all timber species.M. utilis were classified as durable timbers under Malaysian grading rules (Lim et al. 1998), yielded significantly more extractive (7,00%) than the moderately durable (A.laevis) (3,44%) and non-durable (E.malaccense) (0,72%) timbers, in MeOH use the shaker method.The same pattern also occurred when EtOH (5,21%; 2,98% and 0,28% respectively) and acetone (3,25%; 1,67% and 0,44% respectively) were used.However, A. laevis yielded more (1,29%) compared to M. utilis (0,13%) and E. malaccense (0,30) when PETETHR was used.As indicated by several authors (Chang et al. 2001, Syofuna et al. 2012, Ogunwusi et al. 2013), extracts which are low molecular weight compounds in the wood can be extracted by many solvents.However, they differ among timber species, between individual tree of the same species and solvents (Scheffer andCowling 1966, Nacimento et al. 2013) due to genetic variation and environmental (Ericsson et al. 2001).Furthermore, solvent polarity plays a key role in determining the extract yields to be obtained (Bashash et al. 2012).On the other hand, shaker method is not a good method with respect to extraction efficiency for plant materials.As indicated by a few studies before (Park et al. 2001, Kothari et al. 2012), a heat-employing methods' (soxhlet) proved to be the best option for extraction any plant materials.
In another study, even though MeOH is indeed the most common and effective solvent, it has been reported is an environmental pollutant and more toxic than other alcohols (Kapasakalidi et al. 2006, Bridgers et al. 2010).Thus EtOH is preferred as solvent extraction (Delgado-Vargas and Paredes-Lopez 2002).Note: Means with the same letters are not significantly different at 95% confidence limit.

Termite mortality
Among the three timber species tested, M. utilis heartwood samples were very resistant to attack by both subterranean termite; C. gestroi and C. curvignathus.Daily assessment shows that majority of the termite in M. utilis test bottles died within 15 days compared to 18 days in A. laevis and more longer (26 days) in E. malaccense test bottles.Result from this study shows that all castes of termite used (workers and soldiers) either in C. gestroi or C. curvignathus completely died in all M. utilis extracts.A. laevis extracts using PETETHR showed 1,0% and 0,05% while E. malaccense extracts using MeOH showed 0,08% and 0,02% of workers surviving for C. gestroi and C. curvignathus, respectively.Surprisingly, E. malaccense EtOH extracts showed 1,22% of C. gestroi workers surviving at the end of the test.All termites also 100% died when tested with unextracted samples of M. utilis and A. laevis but showed minimal survival (0,01% against C. gestroi and 0,005% against C. curvignathus, respectively) in unextracted E. malaccense samples.Higher survival occurred in brasiliensis test bottles (2,1% against C. gestroi and 2,8% against C. curvignathus).Even though the mortality percentages due to the used wood of extracts were not significantly different from control (brasiliensis), suggesting the toxic effect of these three timber species wood extracts against termites.
The results of termite mortality could be the reaction of termites to the toxic, anti-feeding and / or repellent effects (Yuan and Hu 2011).In all cases, higher termite mortality exists when exposed to un-extracted samples compared to extracted indicates the function of heartwood extractives as a natural wood preservative against termites (Syofuna et al. 2012, Tascioglu et al. 2012, Kirker et al. 2013).In other words, wood extracts are one of the factors that increase the termite mortality.
On the other hand, the results, evidently indicate that M. utilis heartwood extractives contain biologically active compounds that were potent to C. gestroi and C. curvignathus.MeOH apparently was a better solvent in extracting the toxic chemical compounds followed by EtOH, acetone and PETETHR.M. utilis extract completed the mortality of C. gestroi and C. curvignathus at the concentration of 4% compared to A. laevis and E. malaccense even though there was not much difference between C. curvignathus and C. gestroi mortality in every concentration.MeOH extracts that killed both termite species tested may have reacted and made a food substrate to be toxic to both termite species.Golpayegani et al. (2014) in their study on mulberry wood (Morus alba) extractives against Reticulitermes flavipes also found that MeOH is the second best solvent besides acetone that give a low termite survival.On the other hand, Syofuna et al. (2012) reported that different compounds obtained from different solvents will show a different effect towards termite resistance.In conclusion of Taylor et al. (2006) study, to understand the natural durability of wood against termites, we can't just focus on a single compound alone, but this resistance is a combination of several compounds that are present in the wood.Columns with the same letter are not significantly different at α = 0,05.
Toxic effects of three selected..: Kadir  Columns with the same letter are not significantly different at α = 0,05.
In all cases, a significant effect was observed on the weight loss of wood blocks against C. gestroi.All solvents (MeOH, EtOH, acetone and PETETHR) lead to similar trends on the two termites tested (except M. utilis against C. gestroi), probably indicating the presence of the same molecules.Both species of termites also show a similar trend in wood weight loss.However, the performance of wood durability for each timber species depends on the type of solvent used.As reported by González-Laredo et al. (2015), both quantity and particularly the quality of extractives have a key role, but their relative contribution varies considerably from substrate to substrate.On the other hand, studies have shown that extracts of wood is the important factor that determine the durability of wood.This agreed with the study done by Roszaini and Hale (2012) on twelve species of tropical timbers against C. curvignathus and C. gestroi.They found that timbers with high extractive content had high termite resistance and species with lower extractive content showed poor performance.In addition, Lapornik et al. (2005) reported that different solvent extracts different chemical compound.The differences also could be due to the properties of the phenolic components of the plants concerned.

Antitermitic bioassay
The result of antitermitic activities of wood extract is depicted in Table 4, Table 5 and Table 6.The antitermite functions were dependent on the chemical composition of the wood extracts.Previous study indicates that M. utilis extracts under varied concentrations inhibit termite feeding.Similarly, M. utilis extracts showed the strongest anti-termitic activities against C. gestroi and C. curvignathus (Roszaini et al. 2014).It is evident from an earlier study that constituents of wood extracts could affect their anti-termite activity; some influenced greater potency while some others lower, e. g.Monoterpene hydrocarbon possessed lower anti-termite activity as compared with oxygenated constituents (Watanabe et al. 2005, Roszaini et al. 2014).Table 4, Table 5 and Table 6 indicated that heartwood extracts of M. utilis had more anti-termitic activities against both subterranean termites; C. gestroi and C. curvignathus than A. laevis and E. malaccensis.The percentage of paper consumption was 1,01% (MeOH extracts), 1,37% (EtOH extracts), 1,92% (PETETHR extracts) and 1,68% (acetone extracts) at the concentration of 4% against C. gestroi while it was 1,15%; 2,24%; 2,11% and 1,72% respectively, for A. laevis and 1,66%; 2,34%; 2,38% and 2,27%; respectively, for E. malaccensis.The same trend (M.utilis extracts) also occurs on tests against C. curvignathus.Table 4, Table 5 and Table 6 reveal that there was a significant increase in the number of termites in contact with the solvent control disc in comparison to the number of termites on the corresponding extract-treated disc (P<0,05; DF =5) for all wood extracts tested.The current study demonstrated that all four concentrations of the heartwood extract from three different timber species were less preferred and avoided by the both subterranean termite, C. gestroi and C. curvignathus.Lower percentage of paper consumption was obtained at the highest concentration (4%) of every solvent extracts compared to 0,5% of the minimum concentration, respectively.This trend is the same for both termite species Toxic effects of three selected..: Kadir tested.According to the statistical analyses, lower concentration levels (0,5%) of all timber extracts resulted in significant reductions in weight loss when compared to the untreated controls.
Extracts from all timber tested strongly inhibited termite feeding against C. gestroi and C. curvignathus although E. malaccensis heartwood extracts were much less than M. utilis and A. laevis extracts.M. utilis heartwood extracts with MeOH solvents is the most active against both termite species at any level of concentration.At 0,5% level of concentration of any solvent used, M. utilis extracts inhibited ~ 1,5-fold (against C. gestroi) and ~ 1,3-fold (against C. curvignathus) than A. laevis the heartwood extracts and ~ 2,0-fold and ~ 2,3-fold than E. malaccensis, respectively.At the highest level of concentration (4,0%) also in any solvent used, M. utilis extracts inhibited ~ 1,3-fold (against C. gestroi) and ~ 1,5-fold (against C. curvignathus) than A. laevis heartwood extracts and ~ 1,8-fold and ~ 2,0-fold than E. malaccensis, respectively.The strong feeding inhibition from M. utilis heartwood extractives could be due to the higher value of monoterpenes and sesquiterpenes groups where both are interfering with basic behavioural functions of insects (Werner andIllmann 1994, Roszaini et al. 2014).As indicated by other studies, the presence of flavonoids (Ohmura et al. 2000, Wang et al. 2004) and quinones (Nacimento et al. 2013) which possess natural repellent and toxic properties will also increase the durability of the timber against termites.
Findings suggested that all wood extracts may produce larvicidal effects (behaving like general toxicants) against both termites; C. gestroi and C. curvignathus but depends on the solvents used.Laboratory bioassay of M. utilis against both subterranean workers showed that the LC 50 value of C. gestroi was higher than C. curvignathus in all solvents used.Numerical LC 50 values differed based on solvent used (MeOH>EtOH>Acetone>PETETHR).The LC 50 of M. utilis heartwood MeOH extracts was 8,86% for C. gestroi and 8,51% for C. curvignathus, 9,17% and 8,98% for EtOH extracts 10,24% and 10,01% for PETETHR and 9,79% and 9,50% for acetone extracts, respectively (Table 4).Same trend also exists in E. malaccensis except with PETETHR solvents.The LC 50 for E. malaccensis was 10,35% for C. gestroi and 9,88% for C. curvignathus when using MeOH as a solvent 11,42% and 10,85% for EtOH, 11,14% and 11,66 for PETETHR and 13,31% and 12,67% for acetone, respectively (Table 6).Contrarily, with M. utilis and E. malaccensis, the LC 50 result of A. leavis heartwood extract against C. curvignathus was higher than C. gestroi (except when using MeOH solvents).The LC 50 values of MeOH solvents were 9,79% for C. gestroi and 9,15% for C. curvignathus, 10,34% and 11,98% for EtOH, 11,52% and 11,88% for PETETHR and 9,82% and 10,11% for acetone, respectively (Table 5).The lowest of LC 50 values of MeOH extracts followed by EtOH and acetone compared to PETETHR solvents could be to the phenolic content that they extracted.Phenolic content is one of the chemical constituents that influenced the rate of degradation.The higher the total phenolic content, the higher resistivity of the wood species.This means that only low concentrations necessary to turn off at least 50% of the number of termites (Shanbhag and Sundararaj 2013).

Figure 1 .
Figure 1.Weight loss of M. utilis extracted in selective solvents and exposed for 4 weeks to A: C. gestroi and B: C. curvignathus blocks that were subjected to various extraction procedures.Columns with the same letter are not significantly different at α = 0,05.

Figure 2 .
Figure 2. Weight loss of A. laevis extracted in selective solvents and exposed for 4 weeks to A: C. gestroi and B: C. curvignathus of blocks that were subjected to various extraction procedures.Columns with the same letter are not significantly different at α = 0,05.

Figure 3 .
Figure 3. Weight loss of E. malaccensis extracted in selective solvents and exposed for 4 weeks to A: C. gestroi and B: C. curvignathus of blocks that were subjected to various extraction procedures.Columns with the same letter are not significantly different at α = 0,05.

Table 2 .
Mean extractive retentions (mg/m 3 ) in treated filter paper as calculated by solution uptake.

Table 3 .
Effect of different solvent extraction on extractive yields of three wood species.

Table 4 .
Effect of M. utilis wood extracts on feeding and mortality of C. gestroi and C. curvignathus.C. gestroi, CC = C. curvignathus.Con.=Concentration.Mean (+ SD) of 5 replicates for each species.Percentage values followed by the same letter are not significantly different in the same group at the 0,05 level of probability.LC 50 = Lethal Concentration which causes a 50% reduction in feeding as compared to the nontreated control.Maderas.Ciencia y tecnología 19(4): 417 -432, 2017Toxic effects of three selected..: Kadir

Table 5 .
Effect of A. laevis wood extracts on feeding and mortality of C. gestroi and C. curvignathus.

Table 6 .
Effect of E. malaccensis wood extracts on feeding and mortality of C. gestroi and C. curvignathus.