A r t i c l e
TRITERPENE ACIDS FROM APPLE PEEL INHIBIT LEPIDOPTERAN LARVAL MIDGUT LIPASES AND LARVAL GROWTH John T. Christeller The New Zealand Institute for Plant & Food Research, Palmerston North, New Zealand and Institute of Plant Sciences and Resources, Okayama University, Kurashiki, Okayama, Japan
Tony K. McGhie The New Zealand Institute for Plant & Food Research, Palmerston North, New Zealand
Joanne Poulton and Ngaire P. Markwick The New Zealand Institute for Plant & Food Research, Auckland, New Zealand
Fruit extracts from apple, kiwifruit, feijoa, boysenberry, and blueberry were screened for the presence of lipase inhibitory compounds against lepidopteran larval midgut crude extracts. From 120 extracts, six showed significant inhibition with an extract from the peel of Malus × domestica cv. “Big Red” showing highest levels of inhibition. Because this sample was the only apple peel sample in the initial screen, a survey of peels from seven apple cultivars was undertaken and showed that, despite considerable variation, all had inhibitory activity. Successive solvent fractionation and LC-MS of cv. “Big Red” apple peel extract identified triterpene acids as the most important inhibitory compounds, of which ursolic acid and oleanolic acid were the major components and oxo- and hydroxyl-triterpene acids were minor components. When ursolic acid was incorporated into artificial diet and fed to Epiphyas postvittana Walker (Tortricidae: Lepidoptera) larvae at 0.16% w/v, a significant decrease in larval weight was observed Subject Area: nutrition/lipids Grant sponsor: New Zealand Government Public Good Science Fund; Grant number: FRST C06X0804; Grant sponsor: Japan Society for the Promotion of Science (JSPS) Fellowship (to J.T.C.); Grant sponsor: L13549. Correspondence to: John Christeller, Plant and Food Research Institute, Private Bag 11600, Palmerston North 4442, New Zealand. Tel: +64-6953-7666; E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 86, No. 3, 137–150 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21157
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after 21 days. This concentration of ursolic acid is less than half the C 2014 Wiley concentration reported in the skin of some apple cultivars. Periodicals, Inc. Keywords: lipase; triterpene; lepidoptera; ursolic acid; phyllophagy; bioassay
INTRODUCTION Lepidopteran larvae are generally voracious eaters with relative growth rates exceeding 1.0 (g/g)/day (Slansky and Scriber, 1985). The associated high nutritional requirements mean that these larvae, in addition to quantity, must obtain as high quality a diet as possible. In practice this means efficiency at extracting critical nutrients and as much energy as feasible from any given diet, particularly nutritionally poor diets such as leaves. While protein and carbohydrate digestion has been well studied and the enzymes systems characterized (e.g., Markwick et al., 1998), our understanding of lipid digestion remains less well understood. For example, artificial diets (Suckling et al., 1996) for phyllophagous insects generally need supplementation with long chain polyunsaturated fatty acids (LCPUFA). Because lipids are energy-rich compounds they are almost certainly utilized nutritionally by lepidopteran larvae. Lepidopteran larvae have an essential requirement for the LCPUFAs, linoleic and linolenic acids (Dadd, 1985). The absence of these compounds leads to failure of pupal and adult ecdysis and wing deformities in any emerged adults (Canavoso et al., 2001). Recently, detrimental effects on growth, development, and survival of lepidopteran larvae on an artificial diet containing the broad-spectrum lipase inhibitor, tetrahydrolipstatin, have confirmed the requirement for midgut lipases for a lepidopteran larvae feeding on complex lipids (Markwick et al., 2011) and extracts of apple peel showed to be inhibitory of lipase activity (McGhie et al., 2012). These results suggest that lipase inhibitors could act as a resistance mechanism for plant defence, analogous to plant defence mounted against utilization of the major nutritionally important food constituents, proteins, and carbohydrates by the presence of proteinase inhibitors and amylase inhibitors. These LCPUFAs must be obtained from the diet, largely from more complex lipids by hydrolysis using lipases secreted into the midgut lumen. Recent studies (Christeller et al., 2011) have shown that the major lipase activity in phyllophagous lepidopteran larvae are galactolipases (neutral lipase superfamily proteins), able to degrade the major lipids of leaves, the galactolipids. In other dietary classes of lepidopteran larvae, such as granivores, carnivores, and keratinophages, where the major source of long-chain polyunsaturated fatty acids are triacylglycerols (TAGs), TAG lipases (acid lipase superfamily proteins) predominate (Christeller et al., 2011). In the phyllophagous lightbrown apple moth larvae, Epiphyas postvittana Walker (Tortricidae, Lepidoptera), lipase gene expression is very sensitive to lipid quality and quantity (Christeller et al., 2010). Interestingly, these responses are not shown, at the protein level, by the other phyllophagous larva used in that ¨ study, Helicoverpa armigera Hubner (Noctuidae, Lepidoptera) (Christeller et al., 2011). There are numerous reports of secondary metabolite compounds in plants that act as lipase inhibitors (LI) (Hatano et al., 1997; Yamamoto et al., 2000; Han et al., 2001; Shin et al., 2003; Ninomiya et al., 2004; Shin et al., 2004; Xu et al., 2005; Yoshizumi et al., 2006; Karu et al., 2007; Won et al., 2007; Jang et al., 2008; Xie et al., 2008; Kim et al., 2009; Morikawa et al., 2009; Yamada et al., 2010). The mechanism of action of these LIs has not been well studied. There are also rare reports of lipase-inhibitory proteins of vertebrate Archives of Insect Biochemistry and Physiology
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pancreatic lipases (PL). These proteins, soybean lipoxygenase (Satouchi et al., 1998) and soybean ß-amylase (Satouchi et al., 2002) are inhibitory by indirect means, perhaps by modifying the water-membrane interface where lipases are active (Delorme et al., 2011). The inhibitory activity of these proteins may be fortuitous rather than evolved by physiological requirements, especially since they both have well-defined catalytic activities. All these studies have been based on inhibition of vertebrate pancreatic lipases (neutral lipase superfamily proteins) with TAG lipase activity, many with the aim of understanding the antiobesity effects of specific herbal plants. Inhibition of vertebrate gastric lipases (acid lipase superfamily) has been overlooked. We are also unaware of any studies that have used insect digestive lipases to report the presence of LIs, despite the possibility that these compounds could have evolved for this function. Since the preponderance of activity in the midgut of phyllophagous lepidopteran larvae is due to galactolipase activity not TAG lipase activity, these studies may not have identified chemicals that target these specific lipases. Thus, previous studies may not have identified insect-active inhibitory compounds. We report a study in which we screened for LIs in the fruit of common horticultural plants. The concentration of suitably identified compounds could be increased, if necessary by conventional breeding methods if natural variation occurs, to provide a defence against insect pests. We further tested a LI that we identified in this study, the triterpene acid ursolic acid, in artificial diet fed to lepidopteran larvae, for effects on growth, development, and survival.
MATERIALS AND METHODS Fruit Extracts Lipase enzyme and inhibition activities were measured in a library of 120 fruit extracts that had been compiled over a number of years. The methods used to prepare the extracts varied but mostly quantities of fruit were extracted with ethanol (5× ethanol (ml) to fruit (g)) and dried to a powder. For assays the fruit extracts were dissolved in dimethylsulfoxide (DMSO) at 10 mg/ml. Extracts were prepared from a range of cultivars of apple (Malus × domestica), kiwifruit (Actinidia spp.), feijoa (Acca sellowiana), boysenberry (Rubus ursinus × idaeus), and blueberry (Vaccinium spp.) fruit at differing maturities. Preparation of Insect Midgut Lipase Extracts Larvae of H. armigera were raised on artificial diet (McManus and Burgess, 1995) or on tomato leaves. Diet did not affect the lipase levels (Christeller et al., 2011). Fifth instar larvae were dissected and the intact midgut stored at −20◦ C until further processed. One gram of midgut was ground in 5 ml 5 mM HEPES, pH 9.0 containing 2 mM sodium diethyldithiocarbamate, 2 mM isoascorbate, and 2 mM cysteine. The extract was centrifuged and the aliquots of the supernatant were snap frozen at −80◦ C and freeze-dried, then stored at −20◦ C. Lipase activity in the intact midgut and in freeze-dried aliquots was stable for at least 6 months at −20◦ C. For assay, aliquots were reconstituted to the original concentration with ice-cold water and used immediately. The large size of the H. armigera larvae allowed us to prepare suitable amounts of lipase extracts for the broad screening of numerous fruit extracts. Smaller amounts of midgut extract were prepared (as above) from the smaller larvae of a second species, E. postvittana. Archives of Insect Biochemistry and Physiology
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Screen of Fruit Extracts Lipase enzyme activity and inhibition were assayed based on a previously well-established method (Nilsson-Ehle and Schotz, 1976). A stock substrate solution was prepared with 10 g glycerol, 600 mg triolein (Sigma Chemical Co.), and 100 mg whey protein isolate (mostly β-lactoglobulin, A. Hardacre, Department of Food Technology, Massey University, Palmerston North, New Zealand) as the emulsifier. Whey protein gave higher activities than a range of other emulsifying agents tested. The solution was mixed using an UltraTurrax (IKA-Werk, Staufen, Germany) for 15 sec. It could be stored at 4◦ C for extended periods as a clear stable solution. To assay lipase activity, a substrate mix was prepared daily using 250 μl stock substrate solution, 800 μl 50 mM HEPES, pH 8.0, and 2 μl [9, 10 3 H(N)]-triolein (0.5 mCi/ml, Amersham, UK) and sonicated for 15 s/ml. This mix forms an opaque emulsion, stable for several hours. The assay itself comprised 100 μl substrate mix and 100 μl enzyme solution, vortexed, and incubated at 37◦ C for 40 min. To test for the effect of extracts, 10–40 μl aliquots in solvent were added to the substrate emulsion and vortexed before addition of enzyme, that is, the “poisoned interface” method (Gargouri et al., 1991). Control reactions contained an equal amount of solvent. The reaction was stopped and radioactive oleic acid was extracted with 3 ml heptane/chloroform/methanol (1.0/1.25/1.11) and 1 ml 0.1 M sodium carbonate/0.1 M boric acid pH 10.5 then added. After vigorous vortexing for 15 sec, the mixture was allowed to separate into 2 phases. One milliliter of the upper aqueous phase was counted in a scintillation counter. Extract was added to the control reaction after the organic solvent mixture and before the alkaline solution to account for any color quenching during counting. The assay was linear to 20% substrate hydrolyzed. Under these conditions, galactolipases as well as TAG lipases will hydrolyze the TAG substrate (Frederick Carriere, pers. comm.). Purification of Lipase Inhibitors from an Apple Peel Extract The ethanol extract of M. domestica cv. ‘‘Big Red’’ was fractionated by column chromatography using a column containing 2 g Strata X (Phenomenex, Auckland, New Zealand) that had been conditioned with 50 ml methanol followed by 50 ml MilliQ water. ‘‘Big Red’’ fruit extract (95 mg) was suspended in 1.0 ml methanol and applied to the Strata X column. The column was successively eluted with 50 ml aliquots of methanol/water (10/90); methanol/water (30/70); methanol/water (50/50); methanol/water (70/30); methanol, ethyl acetate; and dichloromethane. Each fraction was evaporated to dryness and dissolved in DMSO prior to testing for the presence of lipase inhibitors using the assay described above. Compounds in the most inhibitory fraction were isolated using semipreparative HPLC. All 12 compounds were tested for inhibitory activity (Table 2) and the identity of the most active compounds established by LC-MS analysis (McGhie et al., 2012), with verification using authentic standards where possible. Authentic standards were also assayed using the above radioactivity assay to confirm their lipase inhibitory effect. Effects of Ursolic Acid on Larval Growth and Development In a preliminary experiment, leafroller assay diet (LRA) was prepared as described in Suckling et al. (1996). Six treatments, each of 50 ml diet, were prepared by adding DMSO (Sigma-Aldrich, St Louis, MO) at 1% (0.5 ml), 2, 3, 4, and 5% (2.5 ml) to Archives of Insect Biochemistry and Physiology
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49.5, 49, 48.5, 48, 47.5 ml warm LRA diet (45◦ C), respectively. The control was 50 ml LRA (no DMSO). The diets were poured into Petri dishes and allowed to cool and condition (lose excess water). After 24 h, ∼0.5 ml diet was pushed into the bottom of each 4 ml autoanalyser (AA) cup. Neonate larvae (hatched in the previous 24 h from eggs sterilized in 5% formaldehyde) were weighed in batches of five. Twenty-five larvae per treatment were allowed to feed on diet for 21 days at 21 ± 1◦ C and their survival was monitored. Ursolic acid powder (Qingdao Fraken International Trading Co. Ltd, Qingdao City, Shandong, China; 98% purity) was dissolved in 2 ml DMSO at two concentrations −8 and 2% (i.e., 160 and 40 mg in 2ml DMSO). LRA diet was prepared (as above) and ursolic acid, dissolved in DMSO at two concentrations, was added in 2 ml aliquots to 98 ml LRA (i.e., 2% diet as above) to produce diets containing 0.16 and 0.04% ursolic acid. Two controls, DMSO and water, were prepared by adding 2 ml DMSO and 2 ml sterile (RO) water, respectively, to 98 ml LRA diet. The 0.16% ursolic acid diet was the highest concentration we could achieve given relative solubilities in several different solvents with differing toxicities to the larvae (data not shown). The diets were prepared as above. Neonate E. postvittana, as above, were weighed in batches of five and then transferred individually into the AA cups, 20 per treatment, in three blocks each of four treatments. Larvae were maintained at 21 ± 1◦ C. Survival was recorded weekly and larvae were weighed 21 days after ingesting these diets. Pupation time, and weight and sex of pupae were recorded. E. postvittana larvae were considered better candidates for this experiment, where resources were limited, since they are considerably smaller than H. armigera larvae, and consequently ate less diet.
Statistical Analysis During the period of exponential growth of lepidopteran larvae, plots of logarithms of the weights against time form a straight line. Thus larval growth rate over a defined time period during exponential growth is calculated as loge (final weight) − loge (initial weight) (Markwick et al., 1995). Comparisons of mean weights of both larvae and pupae, and mean larval growth rates were analyzed using a one-way analR ysis of variance followed by multiple comparisons using the Tukey method (Minitab version 16).
RESULTS Inhibition by 120 Fruit Extracts Screening of 120 fruit extracts as described in the methods produced a single extract with marked inhibitory effects and five extracts with a lesser inhibitory effect (data not shown). The former was an extract from the peel of apple fruit (M. × domestica cv. “Big Red”), and was selected for further study. This was the solitary peel extract in the collection we screened. Assays of this extract using E. postvittana larvae, confirmed its inhibitory properties against this second lepidopteran species (data not shown). No further studies were conducted on the other five extracts.
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Table 1. Inhibition of lipase activity by successive fractionation of “Big Red” apple peel. An ethanol-soluble fraction of powdered peel was fractionated and assayed as described in the methods. The results are the mean and standard error of two determinations of the same extracts % Control Rate ± SEM
Fraction Unfractionated 10% methanol 30% methanol 50% methanol 70% methanol 100% methanol 100% ethyl acetate 100% dichloromethane
39.0 76.7 94.1 102.1 104.0 18.1 69.7 90.5
± ± ± ± ± ± ± ±
7.5 5.4 7.9 2.1 0.5 0.3 2.9 1.9
Figure 1. The reversed-phase HPLC chromatogram of the components of apple skin that elute from the SPE with 100% methanol fraction (see Table 1). This fraction contained the majority of lipase inhibitory substances found in cv. “Big Red” apple peel (see Tables 1 and 2). The peaks are labeled according to Table 2 viz 6, hydroxyl-ursolic acid; 10, hydroxyl-ursolic acid; 4, oxo-ursolic acid; 9, coumaroyloxy-ursolic acid; 2, oleanolic acid; 8, ursolic acid. The unlabeled peaks are unidentified, lipase noninhibitory compounds.
Fractionation of M. x Domestica cv. “Big Red” Apple Peel Extract The ethanol extract of ‘‘Big Red’’ apple was fractionated by column chromatography and the inhibition of lipase activities for each of the fractions are shown in Table 1. Analysis by HPLC with diode array detection showed that each fraction contained at least 3–6 major peaks and that the profiles (data not shown) were distinct from each other. The results of the lipase assay (Table 1) show that the largest inhibition occurred with the 100% methanol fraction, with constituent solubilities between 70 and 100% MeOH. Analysis and Identification of Lipase Inhibitors in “Big Red” Methanol Fraction The HPLC chromatogram trace of the “Big Red” 100% methanol fraction is presented as Fig. 1 and using preparative HPLC, 12 compounds were isolated in sufficient amounts for use in the lipase inhibition assay; the results are shown in Table 2. LC-MS analysis of the major inhibitory compounds identified them as ursolic acid, oleanolic acid, and hydroxyl-, oxo-, and coumaryl- derivatives of either ursolic or oleanolic acid (Table 2). The relative inhibitory effects could not be established since the amounts in each fraction were not quantified. However an inhibition assay using authentic ursolic acid indicated a LC50 of < 10 μM. Archives of Insect Biochemistry and Physiology
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Table 2. Inhibition by individual compounds in the 100% methanol fraction separated and isolated by preparative HPLC (see Fig. 1). The data are the results of one of three experiments Compound no. 1 2 3 4 5 6 7 8 9 10 11 12
Tentative IDa
% Control rate
– Oleanolic acid – Oxo-ursolic acid – Hydroxyl-ursolic acid – Ursolic acid Coumaroyloxy-ursolic acid Hydroxyl-ursolic acid – –
92.6 56.1 71.5 58.9 87.2 28.5 79.5 17.3 67.3 43.4 77.7 102.7
a
Tentative refers only to the position of the hydroxyl-, oxo- and coumaroyloxy- substituents on ursolic acid being unassigned. Both ursolic acid and oleanolic acid were confirmed using authentic standards.
Table 3. Inhibition of lipase activity by peel extracts of seven apple cultivars. All peel extracts were prepared as DMSO extracts at 10 mg/mL and assayed as described in the methods Cultivar Envy Fuji Pink Lady Big Red Pacific Rose Granny Smith Braeburn
% Control rate 49.3 40.2 29.5 36.4 35.9 29.1 60.9
Inhibition by Apple Peel Extracts from Seven Cultivars To investigate the distribution of triterpene acids in apple, peel extracts were prepared from six additional apple cultivars and compared to inhibition by the M. x domestica cv. “Big Red” peel extract used in the earlier studies (Table 3). All showed moderate inhibition of lipase activity, extracts from cv. “Braeburn” and cv. “Envy” being less inhibitory whereas those from cv. “Granny Smith” and cv. “Pink Lady” were slightly more inhibitory. Effects of Ursolic Acid on Larval Growth and Development We found it difficult to produce an artificial diet containing homogeneously distributed ursolic acid without adding ursolic acid as a solution in DMSO. Therefore, prior to studies on the effect of ursolic acid on E. postvittana larvae, we assessed the effects of DMSO itself on larval growth and development. The initial mean weights of neonate larvae were not significantly different between treatments (F(5,24) = 0.23, P = 0.948) but at 21 days differences between treatments were significant (F(4,78) = 14.49, P < 0.001). There was no significant difference in mean larval weight between larvae fed for 21 days on control diet and diet containing 1% DMSO, but there was a significant difference in weight between larvae fed on control diet and diet containing 2% DMSO (Tukey, P < 0.05). However, amongst those larvae that pupated, there was no significant weight difference Archives of Insect Biochemistry and Physiology
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Table 4. Mean (± SEM) weights (mg) of Epiphyas postvittana larvae as neonates, and after feeding for 21 days on artificial diets containing DMSO and ursolic acid. Growth rates (calculated as loge 21 day weight – loge initial weight) are also shown. (n) = the number of larvae surviving to 21 days from the original 60 neonates transferred onto the treatments Mean larval weight ± SEM (mg) Treatments 1. 2. 3. 4.
No-additive control DMSO control 0.04% ursolic acid 0.16% ursolic acid
Neonate 0.189 0.192 0.180 0.194
± ± ± ±
0.006a 0.005a 0.008a 0.006a
21 days old 19.53 10.73 10.05 8.05
± ± ± ±
1.10a 0.50b 0.66b,c 0.48c
Growth rates ± SEM (n) 4.52 3.96 3.87 3.63
± ± ± ±
0.08 (52)a 0.05 (54)b 0.09 (48)b,c 0.07 (50)c
Means within a column followed by different superscript letters are significantly different (Tukey: P < 0.05).
amongst either male or female pupae on control diets or on diets containing DMSO at 1 or 2%, indicating that the earlier effects of DMSO at these concentrations, on survival and weight, had disappeared by pupation. Because 2% was the lowest concentration of DMSO that would satisfactorily dissolve ursolic acid and because the effects of 2% DMSO were similar to those of 2% ethanol used for our study with tetrahydrolipstatin (Markwick et al., 2011), we used 2% DMSO in subsequent experiments. There were no significant differences in mean (± SEM) neonate weight (mg) between the three blocks (replicates) within each treatment (P > 0.5) or between the four treatments (F3,47 0.91, P = 0.444) (Table 4). However, after larvae had fed for 21 days on control and treatment diets containing DMSO with or without ursolic acid, there were significant differences in mean larval weight between treatments (although not between blocks within each treatment, that is, no replicate effect). The difference in mean larval weight between the control with no additives and the control with 2% DMSO added was significant (Tukey, P < 0.05), that is, the DMSO additive had a significant effect on the growth of the larvae (Table 4). Mean larval weight at 21 days on the diet containing 0.04% ursolic acid was lower than that on diet containing the DMSO solvent alone but this was not statistically significant (Tukey, P > 0.05). However, both the mean weight and growth rate of larvae fed for 21 days on diet containing 0.16% ursolic acid were significantly lower than those for both the no-additive and DMSO control treatments (Table 4). Thus ursolic acid at 0.16% has a significant effect on the growth rate of E. postvittana larvae over the first 21 days, over and above the negative effect of 2% DMSO on larval growth. There was little effect on survival however; note (n) the number of survivors in each treatment to 21 days (Table 4). At pupation, because of the sexual dimorphism in size, male and female pupal weights were analyzed separately. Amongst males, weight differences between the treatments were significant (ANOVA: F3,92 = 10.97, P < 0.001). However, when the no-additive control treatment was removed, there was no significant difference in mean pupal weights between the DMSO control and the two ursolic acid treatments (Table 5). Amongst females, ANOVA also indicated a significant difference (F3, 83 = 4.44, P = 0.06) between treatments, but as with the males, mean female pupal weights were not significantly different between the ursolic acid treatments and the DMSO control. The mean female pupal weight was significantly higher in the no-additive control than in the DMSO control and the 0.16% ursolic acid treatment, but not significantly different from the 0.04% ursolic acid treatment (Table 5). Archives of Insect Biochemistry and Physiology
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Table 5. Mean (± SEM) weights (mg) at pupation, and duration of development to pupation (mean ± SEM days) for Epiphyas postvittana larvae fed since neonates on artificial diets containing DMSO and ursolic acid. (n) = the number of larvae surviving to pupation from the original 60 neonates transferred onto the treatments Mean pupal weight ± SEM (mg) Treatments 1. 2. 3. 4.
water control DMSO control 0.04% ursolic acid 0.16% ursolic acid
Male (n) 31.86 27.58 27.95 27.88
± ± ± ±
0.44 (25)a 0.57 (24)b 0.83 (22)b 0.64 (25)b
Female (n) 50.42 48.59 53.49 46.59
± ± ± ±
1.48 (19)ab 1.72 (19)b 1.28 (24)a 1.41 (25)b
Mean days ± SEM to pupation Male 31.40 34.42 33.68 35.42
± ± ± ±
0.48a 0.59b 0.47b 0.66b
Female 35.20 38.77 38.74 41.72
± ± ± ±
0.63a 1.25b 0.98ab 0.91b
Means within a column followed by different superscript letters are significantly different (Tukeys: P < 0.05).
Ursolic acid treatments also increased the time to pupation; the mean number of days to pupation for each treatment is given in Table 5. The mean time to pupation for both males and females was significantly longer in two treatments (DMSO and 0.16% ursolic acid) than in the no-additive control, but there was no significant difference between the DMSO and ursolic acid treatments. These treatments generally showed higher variability in pupal weights.
DISCUSSION The activity-based purification of the active lipase-inhibitory components of a methanolic apple peel extract were found to be triterpene acids; ursolic acid, oleanolic acid, and monohydroxy-, keto- and coumaroyloxy-dervatives of these compounds. The presence of ursolic and oleanolic acids in apple skin (Belding et al., 1998, Frighetto et al., 2008, Andre et al., 2012) is well known and has been previously reported in apple leaves too (Bringe et al., 2006). In both tissues they are contained within the epicuticular wax and form a major component thereof. Our finding (Table 3) that cv. “Fuji” extracts contains higher inhibitory activity than cv. “Granny Smith” is consistent with those of Frighetto et al. (2008) that ursolic acid was higher in cv. “Fuji” than in cv. “Granny Smith.” The data of Andre et al. (2012) indicate dramatic variation, between 0.004 and 0.35% of apple skin is ursolic acid and between 0.004 and 0.08% is oleanolic acid, depending on cultivar. Several authors have reported the effect of ursolic acid and related compounds on PL activity. Jang et al. (2008) reported five triterpene acids (including ursolic acid) from the roots of Actinidia arguta inhibited PL and Kim et al. (2009) reported that ursolic acid from the same source inhibited phosphodiesterase activity. Ninomiya et al. (2004) reported inhibition of PL from Salvia officinalis leaves and inhibition of PL by triterpene glycosides and saponins have also been noted (Morikawa and Yoshikawa, 2004; Xu et al., 2005; Yoshizumi et al., 2006). The mode of inhibition of these compounds is largely unclarified since normal solution kinetics are not applicable to heterogeneous lipase assays. Our data are consistent with these studies but report inhibition from a completely different enzyme source. While earlier studies have focused on inhibition of PL with an interest on dietary supplementation using ursolic acid as an antiobesity factor, our work has used an enzyme preparation that contains galactolipase: TAG lipase activities in a 2.6:1 ratio (Christeller et al., 2011). The extent of inhibition observed indicates that both Archives of Insect Biochemistry and Physiology
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activities are inhibited. Genomic studies (Simpson et al., 2007; Christeller, unpublished data) further show that while lepidopteran midgut galactolipases are members of the pancreatic lipase superfamily, the TAG lipases are members of the gastric lipase superfamily. To our knowledge, no studies showing effects of these compounds on vertebrate gastric lipases have been reported but our results suggest that they are likely to be inhibited. Insect midgut galactolipases and TAG lipases are inhibited not only by ursolic acid but also by oleanolic acid and by some triterpene acid derivatives (Table 2). While we have previously shown that lepidopteran larval midgut lipase activities are sensitive to the lipase inhibitor, tetrahydrolipstatin (Markwick et al., 2011), this is the first report that these lipase activities are inhibited by plant-derived compounds. Our findings (Table 3) that variation in lipase inhibitory activity occurs between cultivars and is consistent with being associated with the triterpene acid components together indicate that the potential to enhance triterpene acid levels in apple skin of elite cultivars exists, either by direct measurement or by determining genetic markers for triterpene acid accumulation. The function(s) of triterpene acids in plants is unclear. They accumulate in diverse forms in the intracuticlular layer (Buschhaus and Jetter, 2011) of many, perhaps most, even all plants. Their diversity is surely attributable to the broad specificity of plant P450s for which they are substrates and their properties may be important to the chemical properties of functioning cuticles (Vogg et al., 2004; Yu et al., 2007). Whether their presence evolved to contribute to pest deterrence or fungal resistance, with many fungi including cutinase and other lipases (Reis et al., 2005; Voigt et al., 2005; Bravo-Ruiz et al., 2013) among their pathological determinants, awaits further studies. Our preliminary bioassay showed that DMSO at a concentration at 2% in diets had a significant effect on the growth and development of E. postvittana larvae. Taking this into account, there was a further significant effect of ursolic acid on larval growth and development in this species: at 0.16%, ursolic acid significantly reduced the larval weight and growth rate during the first 21 day feeding. However, at pupation, mean weights were not significantly different in DMSO treatments with and without ursolic acid, although in all three treatments, mean pupal weights were significantly lower than those in the noadditive control. Time to pupation was delayed in the ursolic acid treatments, although this was also not significantly different from the DMSO control. Survival was not significantly affected by ingestion of ursolic acid at concentrations used in this experiment. Therefore, the main impact of ursolic acid treatments was early in the life-cycle/development of the E. postvittana larvae. It seems likely that neonates are deficient in long-chain polyunsaturated fatty acids but accumulate adequate amounts under the experimental conditions within 3 weeks to restore normal growth rates. The values of 0.04 and 0.16% ursolic acid in diet used in our experiment are at the mid to lower end of ursolic acid values reported in apple skin (Andre et al., 2012)with only two russet varieties of 109 apple varieties measured with values below 0.02% and where up to 2.2-fold higher values (0.35%) were found. Our data, reporting a significant effect on initial growth rates, suggest that ursolic acid might act as a growth inhibitor in some cultivars with high concentration but not in others with low concentrations. Bioassays on intact apple fruit from a range of cultivars may support this conclusion. The behavior of the larvae of E. postvittana might also indicate a role for ursolic acid effects. Eggs are laid on leaves and neonate larvae prefer to feed on shoot tips (Geier and Briese, 1980; Lo et al., 2000). Other leaves or leaf pairs are utilized as the preferred sites are occupied, and feeding on fruit (Geier and Briese, 1980; Lo et al., 2000) or more commonly leaf-fruit pairs, is only observed later in the season when the fruit area is proportionally higher Archives of Insect Biochemistry and Physiology
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(Suckling and Ioratti, 1996). Bringe et al. (2006) noted that the concentration of ursolic acid is lower in leaves and declines rapidly as leaves age. It is probable that larvae feed on fruit only by chance as other feeding sites become scarcer; thus higher concentrations of ursolic acid might deter even this limited feeding activity. Fruit-feeding lepidopteran larvae such as those of Cydia pomonella seem to avoid feeding on the skin by burrowing directly into the center of the fruit. Increasing ursolic acid may also improve fruit health attributes with a plethora of in vitro, cell-based and mouse model studies reported in the literature (e.g., Kang et al., 2008; Jang et al., 2009; Wang et al., 2010; Jang et al., 2010; Kunkel et al., 2011; Rao et al., 2011). Health benefits of triterpenoids have been recently reviewed (Szakiel et al., 2012) but would be, however, contingent on consumers eating the skin of their apples. A breeding program, either by direct analysis or by marker-assisted breeding, for increased triterpene acids in the peel of apple and potentially other fruits, could therefore meet dual objectives of insect feeding deterrence and improved health properties for consumers.
ACKNOWLEDGMENTS We would like to thank Dr. A. Barrington for providing the insects and Dr. D. Rowan and Dr. W. Laing for useful discussions. Neither conflicts of interest, financial or otherwise, nor any breaches of ethical standards were identified by the authors.
Literature Cited Andre CM, Greenwood JM, Walker EG, Rassam M, Sullivan M, Evers D, Perry NB, Laing WA. 2012. Anti-inflammatory procyanidins and triterpenes in 109 apple varieties. J Agriculture Food Chem 60: 10546–10554. Belding RD, Blankenship SM, Young E, Leidy RB. 1998, Composition and variability of epicuticular waxes in apple cultivars. J Amer Soc Hort Sci 123: 348–356. Bravo-Ruiz G, Ruiz-Roldan C, Roncero MIG. 2013. Lipolytic System of the tomato pathogen Fusarium oxysporum f. sp lycopersici. Molec Plant-Microbe Interactions 26: 1054–1067. Bringe K, Schumacher CFA, Schmitz-Eiberger M, Steiner U, Oerke EC. 2006. Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces. Phytochem 67: 161–170. Buschhaus C, Jetter R. 2011. Composition differences between epicuticular and intracuticular wax substructures: How do plants seal their epidermal surfaces? J Exp Bot 62: 841–853. Canavoso LE, Jouni ZE, Karnas KJ, Pennington JE, Wells MA. 2001. Fat metabolism in insects. Ann Review Nutrition 21: 23–46. Christeller JT, Amara S, Carriere F. 2011. Galactolipase, phospholipase and triacylglycerol lipase activities in the midgut of six species of lepidopteran larvae feeding on different lipid diets. J Insect Physiol 57: 1232–1239. Christeller JT, Poulton J, Markwick NM, Simpson RM. 2010. The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth Epiphyas postvittana Walker Tortricidae. Insect Molec Biol 191: 9–25. Dadd RH. 1985. Nutrition, organisms. In: Kerkut GA, Gilbert LI, editors. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Oxford, UK: Pergamon Press. p. 313–390. Delorme V, Dhouib R, Canaan S, Fotiadu F, Carriere F, Cavalier JF. 2011. effects of surfactants on lipase structure, activity, and inhibition. Pharm Res 8: 1831–1842. Archives of Insect Biochemistry and Physiology
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Frighetto RTS, Welendorf RM, Nigro EN, Frighetto N, Siani AC. 2008. Isolation of ursolic acid from apple peels by high speed counter-current chromatograph. Food Chem 106: 767–771. Gargouri Y, Chahinian H, Moreau, H, Ransac S, Verger R. 1991. Inactivation of Pancreatic and Gastric Lipases by THL and C12-o-TNB: A kinetic study with emulsified tributyrin. Biochim Biophys Acta 1085: 322–328. Geier PW, Briese DT. 1980. The light-brown apple moth, Epiphyas postvittana (Walker): 4. Studies on the population dynamics and injuriousness to apples in the Australian Capital Territory. Aust J Ecol 5: 63–93. Han LK, Kimura Y, Kawashima M, Takaku T, Taniyama T, Hayashi T, Zheng YN, Okuda H. 2001. Anti-obesity effects in rodents of dietary teasaponin, a lipase inhibitor. Internat J Obesity 25: 1459–1464. Hatano T, Yamashita A, Hashimoto T, Ito H, Kubo N, Yoshiyama M, Shimura S, Itoh Y, Okuda T, Yoshida T. 1997. Flavan dimers with lipase inhibitory activity from Cassia nomame. Phytochem 46: 893–900. Jang DS, Lee GY, Kim J, Lee YM, Kim JM, Kim YS, Kim JS. 2008. A new pancreatic lipase inhibitor isolated from the roots of Actinidia arguta. Arch Pharmacal Res 31: 666–670. Jang S-M, Yee S-T, Choi J, Choi M-S, Do G-M, Jeon S-M, Yeo J, Kim M-J, Seo K-I, Lee M-K. 2009. Ursolic acid enhances the cellular immune system and pancreatic beta-cell function in streptozotocininduced diabetic mice fed a high-fat diet. Internat Immunopharmacol 9: 113–119. Jang S-M, Kim M-J, Choi M-S, Kwon E-Y, Lee M-K. 2010. Inhibitory effects of ursolic acid on hepatic polyol pathway and glucose production in streptozotocin-induced diabetic mice. Clinical Exp Metabol 59: 512–519. Kang SY, Yoon SY, Roh DH, Jeon MJ, Seo HS, Uh DK, Kwon YB, Kim HW, Han HJ, Lee HJ, Lee JH. 2008. The anti-arthritic effect of ursolic acid on zymosan-induced acute inflammation-induced chronic arthritis models and adjuvant. J Pharmacy Pharmacol 60: 1347–1354. Karu N, Reifen R, Kerem Z. 2007. Weight gain reduction in mice fed Panax ginseng saponin, a pancreatic lipase inhibitor. J Agricultural Food Chem 55: 2824–2828. Kim J, Jang DS, Kim H, Kim JS. 2009. Anti-lipase and lipolytic activities of ursolic acid isolated from the roots of Actinidia arguta. Arch Pharmacal Res 32: 983–987. Kunkel SD, Suneja M, Ebert SM, Bongers KS, Fox DK, Malmberg SE, Alipour F, Shields RK, Adams CM. 2011. mRNA Expression Signatures of Human Skeletal Muscle Atrophy Identify a Natural Compound that Increases Muscle Mass. Cell Metabol 13: 627–638. Lo PL, Suckling DM, Bradley SJ, Walker JTS, Shaw PW, Burnip GM. 2000. Factors affecting feeding site preferences of lightbrown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae) on apple trees in New Zealand. New Zealand J Crop Food Sci 28: 235–243. Markwick NP, Poulton J, McGhie TK, Wohlers MW, Christeller JT. 2011. The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana Walker Tortricidae, Lepidoptera. J Insect Physiol 5712: 1643–1650. Markwick NP, Laing WA, Christeller JT, McHenry JZ, Newton MR. 1998. Overproduction of digestive enzymes compensates for inhibitory effects of protease and alpha-amylase inhibitors fed to three species of leafrollers Lepidoptera, Tortricidae. J Economic Entomol 916: 1265–1276. Markwick NP, Reid SJ, Laing WA, Christeller JT. 1995. Effects of dietary protein and protease inhibitors on codling moth (Lepidoptera: Tortricidae). J Economic Entomol 88: 33–39. McGhie T K, Hudault S, Lunken RCM, Christeller JT. 2012. Apple peels, from seven cultivars, have lipase-inhibitory activity and contain numerous ursenoic acids as identified by LC-ESI-QTOFHRMS. J Agricultural Food Chem 601: 482–491. McManus MT, Burgess EPJ. 1995. Effects of the soybean kunitz trypsin-inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 41: 731–738.
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Morikawa T, Xie YY, Asao Y, Okamoto M, Yamashita C, Muraoka O, Matsuda H, Pongpiriyadacha Y, Yuan D, Yoshikawa M. 2009. Oleanane-type triterpene oligoglycosides with pancreatic lipase inhibitory activity from the pericarps of Sapindus rarak. Phytochem 70: 1166–1172. Nilsson-Ehle P, Schotz MC. 1976. A stable, radioactive substrate emulsion for assay of Iipoprotein Iipase. J Lipid Res 17: 536–541. Ninomiya K, Matsuda H, Shimoda H, Norihisa N, Kasajima N, Yoshino T, Morikawa T, Yoshikawa M. 2004. Carnosic acid, a new class of lipid absorption inhibitor from sage. Bioorg Med Chem Lett 14: 1943–1946. Rao VS, de Melo CL, Queiroz MGR, Lemos TLG, Menezes DB, Melo TS, Santos FA. 2011. Ursolic acid, a pentacyclic triterpene from Sambucus australis, prevents abdominal adiposity in mice fed a high-fat diet. J Med Food 14: 1375–82. Reis H, Pfiffi S, Hahn M. 2005. Molecular and functional characterization of a secreted lipase from Botrytis cinerea. Mol Plant Pathol 6: 257–267. Satouchi K, Hirano K, Fujino O, Ikoma M, Tanaka T, Kitamura K. 1998. Lipoxygenase-1 from soybean seed inhibiting the activity of pancreatic lipase. Biosci Biotechnol Biochem 62: 1498– 1503. Satouchi K, Kodama Y, Murakami K, Tanaka T, Iwamoto H, Ishimoto M. 2002. A lipaseinhibiting protein from lipoxygenase-deficient soybean seeds. Biosci Biotechnol Biochem 66: 2154–2160. Sheng L, Qian ZY, Zheng SG, Xi L. 2006. Mechanism of hypolipidemic effect of crocin in rats, Crocin inhibits pancreatic lipase. Europ J Pharmacol 543: 116–122. Shin JE, Han MJ, Kim DH. 2003. 3-Methylethergalangin isolated from Alpinia officinarum inhibits pancreatic lipase. Biol Pharm Bul 26: 854–857. Shin JE, Han MJ, Song MC, Baek NI, Kim DH. 2004. 5-hydroxy-7–4 ‘-hydroxy-3 ‘-methoxyphenyl1-phenyl-3-heptanone: A pancreatic lipase inhibitor isolated from Alpinia officinarum. Biol Pharm Bul 27: 138–140. Simpson RM, Newcomb RD, Gatehouse HS, Crowhurst RN, Chagne D, Gatehouse LN, Markwick NP, Beuning LL, Murray C, Marshall SD, Yauk YK, Nain B, Wang YY, Gleave AP, Christeller JT. 2007. Expressed sequence tags from the midgut of Epiphyas postvittana Walker Lepidoptera, Tortricidae. Insect Molec Biol 16: 675–690. Slansky FJ, Scriber JM. 1985. Food Consumption and Utilization. In: Kerkut GA, Gilbert LI, editors. Comprehensive Insect Physiology Biochemistry and Pharmacology. Oxford: Pergamon Press. p. 87–163. Suckling DM, Ioratti C. 1996. Behavioral responses of leafroller larvae to apple leaves and fruit. Entomol Exp Appl 81: 97–103. Suckling DM, Markwick NP, Wigley PJ, Frater CM, Chilcott CN, Maindonald JH. 1996. Response to purified Cry 1B protein in lab-wild type crosses of Light Brown Apple Moth. Proceedings of 49th New Zealand Plant Protection Conference 49: 64–70. Szakiel A, Paczkowski C, Pensec F, Bertsch C. 2012. Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochem Rev 11: 263–284. Voigt CA, Schafer W, Salomon S. 2005. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals. Plant J 42: 364–375. Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M. 2004. Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase. J Exp Bot 55: 1401– 1410. Wang ZH, Hsu CC, Huang CN, Yin MC. 2010. Anti-glycative effects of oleanolic acid and ursolic acid in kidney of diabetic mice. Europ J Pharmacol 628: 255–260. Won SR, Kim SK, Kim YM, Lee PH, Ryu JH, Kim JW, Rhee HI. 2007. Licochalcone A, A lipase inhibitor from the roots of Glycyrrhiza uralensis. Food Res Internat 40: 1046–1050. Archives of Insect Biochemistry and Physiology
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Xie WD, Zhang Y, Wang NL, Zhou H, Du LJ, Ma XH, Shi XJ, Cai GP. 2008. Novel effects of macrostemonoside A, a compound from Allium macrostemon Bung, on hyperglycemia, hyperlipidemia, and visceral obesity in high-fat diet-fed C57BL/6 mice. Europ J Pharmacol 599: 159–165. Xu BJ, Han LK, Zheng YN, Lee JH, Sung CK. 2005. In vitro inhibitory effect of triterpenoidal saponins from Platycodi Radix on pancreatic lipase. Arch Pharmacal Res 28: 180–185. Yamada K, Murata T, Kobayashi K, Miyase T, Yoshizaki F. 2010. A lipase inhibitor monoterpene and monoterpene glycosides from Monarda punctata. Phytochem 71: 1884–1891. Yamamoto M, Shimura S, Itoh Y, Ohsaka T, Egawa M, Inoue S. 2000. Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs, Nomame herba, on rats fed a high-fat diet. Internat J Obesity 24: 758–764. Yoshizumi K, Hirano K, Ando H, Hirai Y, Ida Y, Tsuji T, Tanaka T, Satouchi K, Terao, J. 2006. Lupane-type saponins from leaves of Acanthopanax sessiliflorus and their inhibitory activity on pancreatic lipase. J Agricultural Food Chem 54: 335–341. Yu MML, Schulze HG, Jetter R, Blades MW, Turner RFB. 2007. Raman microspectroscopic analysis of triterpenoids found in plant cuticles. Appl Spectrosc 61: 32–37.
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TRITERPENE ACIDS FROM APPLE PEEL INHIBIT LEPIDOPTERAN LARVAL MIDGUT LIPASES AND LARVAL GROWTH John T. Christeller The New Zealand Institute for Plant & Food Research, Palmerston North, New Zealand and Institute of Plant Sciences and Resources, Okayama University, Kurashiki, Okayama, Japan
Tony K. McGhie The New Zealand Institute for Plant & Food Research, Palmerston North, New Zealand
Joanne Poulton and Ngaire P. Markwick The New Zealand Institute for Plant & Food Research, Auckland, New Zealand
Fruit extracts from apple, kiwifruit, feijoa, boysenberry, and blueberry were screened for the presence of lipase inhibitory compounds against lepidopteran larval midgut crude extracts. From 120 extracts, six showed significant inhibition with an extract from the peel of Malus × domestica cv. “Big Red” showing highest levels of inhibition. Because this sample was the only apple peel sample in the initial screen, a survey of peels from seven apple cultivars was undertaken and showed that, despite considerable variation, all had inhibitory activity. Successive solvent fractionation and LC-MS of cv. “Big Red” apple peel extract identified triterpene acids as the most important inhibitory compounds, of which ursolic acid and oleanolic acid were the major components and oxo- and hydroxyl-triterpene acids were minor components. When ursolic acid was incorporated into artificial diet and fed to Epiphyas postvittana Walker (Tortricidae: Lepidoptera) larvae at 0.16% w/v, a significant decrease in larval weight was observed Subject Area: nutrition/lipids Grant sponsor: New Zealand Government Public Good Science Fund; Grant number: FRST C06X0804; Grant sponsor: Japan Society for the Promotion of Science (JSPS) Fellowship (to J.T.C.); Grant sponsor: L13549. Correspondence to: John Christeller, Plant and Food Research Institute, Private Bag 11600, Palmerston North 4442, New Zealand. Tel: +64-6953-7666; E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 86, No. 3, 137–150 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21157
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after 21 days. This concentration of ursolic acid is less than half the C 2014 Wiley concentration reported in the skin of some apple cultivars. Periodicals, Inc. Keywords: lipase; triterpene; lepidoptera; ursolic acid; phyllophagy; bioassay
INTRODUCTION Lepidopteran larvae are generally voracious eaters with relative growth rates exceeding 1.0 (g/g)/day (Slansky and Scriber, 1985). The associated high nutritional requirements mean that these larvae, in addition to quantity, must obtain as high quality a diet as possible. In practice this means efficiency at extracting critical nutrients and as much energy as feasible from any given diet, particularly nutritionally poor diets such as leaves. While protein and carbohydrate digestion has been well studied and the enzymes systems characterized (e.g., Markwick et al., 1998), our understanding of lipid digestion remains less well understood. For example, artificial diets (Suckling et al., 1996) for phyllophagous insects generally need supplementation with long chain polyunsaturated fatty acids (LCPUFA). Because lipids are energy-rich compounds they are almost certainly utilized nutritionally by lepidopteran larvae. Lepidopteran larvae have an essential requirement for the LCPUFAs, linoleic and linolenic acids (Dadd, 1985). The absence of these compounds leads to failure of pupal and adult ecdysis and wing deformities in any emerged adults (Canavoso et al., 2001). Recently, detrimental effects on growth, development, and survival of lepidopteran larvae on an artificial diet containing the broad-spectrum lipase inhibitor, tetrahydrolipstatin, have confirmed the requirement for midgut lipases for a lepidopteran larvae feeding on complex lipids (Markwick et al., 2011) and extracts of apple peel showed to be inhibitory of lipase activity (McGhie et al., 2012). These results suggest that lipase inhibitors could act as a resistance mechanism for plant defence, analogous to plant defence mounted against utilization of the major nutritionally important food constituents, proteins, and carbohydrates by the presence of proteinase inhibitors and amylase inhibitors. These LCPUFAs must be obtained from the diet, largely from more complex lipids by hydrolysis using lipases secreted into the midgut lumen. Recent studies (Christeller et al., 2011) have shown that the major lipase activity in phyllophagous lepidopteran larvae are galactolipases (neutral lipase superfamily proteins), able to degrade the major lipids of leaves, the galactolipids. In other dietary classes of lepidopteran larvae, such as granivores, carnivores, and keratinophages, where the major source of long-chain polyunsaturated fatty acids are triacylglycerols (TAGs), TAG lipases (acid lipase superfamily proteins) predominate (Christeller et al., 2011). In the phyllophagous lightbrown apple moth larvae, Epiphyas postvittana Walker (Tortricidae, Lepidoptera), lipase gene expression is very sensitive to lipid quality and quantity (Christeller et al., 2010). Interestingly, these responses are not shown, at the protein level, by the other phyllophagous larva used in that ¨ study, Helicoverpa armigera Hubner (Noctuidae, Lepidoptera) (Christeller et al., 2011). There are numerous reports of secondary metabolite compounds in plants that act as lipase inhibitors (LI) (Hatano et al., 1997; Yamamoto et al., 2000; Han et al., 2001; Shin et al., 2003; Ninomiya et al., 2004; Shin et al., 2004; Xu et al., 2005; Yoshizumi et al., 2006; Karu et al., 2007; Won et al., 2007; Jang et al., 2008; Xie et al., 2008; Kim et al., 2009; Morikawa et al., 2009; Yamada et al., 2010). The mechanism of action of these LIs has not been well studied. There are also rare reports of lipase-inhibitory proteins of vertebrate Archives of Insect Biochemistry and Physiology
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pancreatic lipases (PL). These proteins, soybean lipoxygenase (Satouchi et al., 1998) and soybean ß-amylase (Satouchi et al., 2002) are inhibitory by indirect means, perhaps by modifying the water-membrane interface where lipases are active (Delorme et al., 2011). The inhibitory activity of these proteins may be fortuitous rather than evolved by physiological requirements, especially since they both have well-defined catalytic activities. All these studies have been based on inhibition of vertebrate pancreatic lipases (neutral lipase superfamily proteins) with TAG lipase activity, many with the aim of understanding the antiobesity effects of specific herbal plants. Inhibition of vertebrate gastric lipases (acid lipase superfamily) has been overlooked. We are also unaware of any studies that have used insect digestive lipases to report the presence of LIs, despite the possibility that these compounds could have evolved for this function. Since the preponderance of activity in the midgut of phyllophagous lepidopteran larvae is due to galactolipase activity not TAG lipase activity, these studies may not have identified chemicals that target these specific lipases. Thus, previous studies may not have identified insect-active inhibitory compounds. We report a study in which we screened for LIs in the fruit of common horticultural plants. The concentration of suitably identified compounds could be increased, if necessary by conventional breeding methods if natural variation occurs, to provide a defence against insect pests. We further tested a LI that we identified in this study, the triterpene acid ursolic acid, in artificial diet fed to lepidopteran larvae, for effects on growth, development, and survival.
MATERIALS AND METHODS Fruit Extracts Lipase enzyme and inhibition activities were measured in a library of 120 fruit extracts that had been compiled over a number of years. The methods used to prepare the extracts varied but mostly quantities of fruit were extracted with ethanol (5× ethanol (ml) to fruit (g)) and dried to a powder. For assays the fruit extracts were dissolved in dimethylsulfoxide (DMSO) at 10 mg/ml. Extracts were prepared from a range of cultivars of apple (Malus × domestica), kiwifruit (Actinidia spp.), feijoa (Acca sellowiana), boysenberry (Rubus ursinus × idaeus), and blueberry (Vaccinium spp.) fruit at differing maturities. Preparation of Insect Midgut Lipase Extracts Larvae of H. armigera were raised on artificial diet (McManus and Burgess, 1995) or on tomato leaves. Diet did not affect the lipase levels (Christeller et al., 2011). Fifth instar larvae were dissected and the intact midgut stored at −20◦ C until further processed. One gram of midgut was ground in 5 ml 5 mM HEPES, pH 9.0 containing 2 mM sodium diethyldithiocarbamate, 2 mM isoascorbate, and 2 mM cysteine. The extract was centrifuged and the aliquots of the supernatant were snap frozen at −80◦ C and freeze-dried, then stored at −20◦ C. Lipase activity in the intact midgut and in freeze-dried aliquots was stable for at least 6 months at −20◦ C. For assay, aliquots were reconstituted to the original concentration with ice-cold water and used immediately. The large size of the H. armigera larvae allowed us to prepare suitable amounts of lipase extracts for the broad screening of numerous fruit extracts. Smaller amounts of midgut extract were prepared (as above) from the smaller larvae of a second species, E. postvittana. Archives of Insect Biochemistry and Physiology
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Screen of Fruit Extracts Lipase enzyme activity and inhibition were assayed based on a previously well-established method (Nilsson-Ehle and Schotz, 1976). A stock substrate solution was prepared with 10 g glycerol, 600 mg triolein (Sigma Chemical Co.), and 100 mg whey protein isolate (mostly β-lactoglobulin, A. Hardacre, Department of Food Technology, Massey University, Palmerston North, New Zealand) as the emulsifier. Whey protein gave higher activities than a range of other emulsifying agents tested. The solution was mixed using an UltraTurrax (IKA-Werk, Staufen, Germany) for 15 sec. It could be stored at 4◦ C for extended periods as a clear stable solution. To assay lipase activity, a substrate mix was prepared daily using 250 μl stock substrate solution, 800 μl 50 mM HEPES, pH 8.0, and 2 μl [9, 10 3 H(N)]-triolein (0.5 mCi/ml, Amersham, UK) and sonicated for 15 s/ml. This mix forms an opaque emulsion, stable for several hours. The assay itself comprised 100 μl substrate mix and 100 μl enzyme solution, vortexed, and incubated at 37◦ C for 40 min. To test for the effect of extracts, 10–40 μl aliquots in solvent were added to the substrate emulsion and vortexed before addition of enzyme, that is, the “poisoned interface” method (Gargouri et al., 1991). Control reactions contained an equal amount of solvent. The reaction was stopped and radioactive oleic acid was extracted with 3 ml heptane/chloroform/methanol (1.0/1.25/1.11) and 1 ml 0.1 M sodium carbonate/0.1 M boric acid pH 10.5 then added. After vigorous vortexing for 15 sec, the mixture was allowed to separate into 2 phases. One milliliter of the upper aqueous phase was counted in a scintillation counter. Extract was added to the control reaction after the organic solvent mixture and before the alkaline solution to account for any color quenching during counting. The assay was linear to 20% substrate hydrolyzed. Under these conditions, galactolipases as well as TAG lipases will hydrolyze the TAG substrate (Frederick Carriere, pers. comm.). Purification of Lipase Inhibitors from an Apple Peel Extract The ethanol extract of M. domestica cv. ‘‘Big Red’’ was fractionated by column chromatography using a column containing 2 g Strata X (Phenomenex, Auckland, New Zealand) that had been conditioned with 50 ml methanol followed by 50 ml MilliQ water. ‘‘Big Red’’ fruit extract (95 mg) was suspended in 1.0 ml methanol and applied to the Strata X column. The column was successively eluted with 50 ml aliquots of methanol/water (10/90); methanol/water (30/70); methanol/water (50/50); methanol/water (70/30); methanol, ethyl acetate; and dichloromethane. Each fraction was evaporated to dryness and dissolved in DMSO prior to testing for the presence of lipase inhibitors using the assay described above. Compounds in the most inhibitory fraction were isolated using semipreparative HPLC. All 12 compounds were tested for inhibitory activity (Table 2) and the identity of the most active compounds established by LC-MS analysis (McGhie et al., 2012), with verification using authentic standards where possible. Authentic standards were also assayed using the above radioactivity assay to confirm their lipase inhibitory effect. Effects of Ursolic Acid on Larval Growth and Development In a preliminary experiment, leafroller assay diet (LRA) was prepared as described in Suckling et al. (1996). Six treatments, each of 50 ml diet, were prepared by adding DMSO (Sigma-Aldrich, St Louis, MO) at 1% (0.5 ml), 2, 3, 4, and 5% (2.5 ml) to Archives of Insect Biochemistry and Physiology
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49.5, 49, 48.5, 48, 47.5 ml warm LRA diet (45◦ C), respectively. The control was 50 ml LRA (no DMSO). The diets were poured into Petri dishes and allowed to cool and condition (lose excess water). After 24 h, ∼0.5 ml diet was pushed into the bottom of each 4 ml autoanalyser (AA) cup. Neonate larvae (hatched in the previous 24 h from eggs sterilized in 5% formaldehyde) were weighed in batches of five. Twenty-five larvae per treatment were allowed to feed on diet for 21 days at 21 ± 1◦ C and their survival was monitored. Ursolic acid powder (Qingdao Fraken International Trading Co. Ltd, Qingdao City, Shandong, China; 98% purity) was dissolved in 2 ml DMSO at two concentrations −8 and 2% (i.e., 160 and 40 mg in 2ml DMSO). LRA diet was prepared (as above) and ursolic acid, dissolved in DMSO at two concentrations, was added in 2 ml aliquots to 98 ml LRA (i.e., 2% diet as above) to produce diets containing 0.16 and 0.04% ursolic acid. Two controls, DMSO and water, were prepared by adding 2 ml DMSO and 2 ml sterile (RO) water, respectively, to 98 ml LRA diet. The 0.16% ursolic acid diet was the highest concentration we could achieve given relative solubilities in several different solvents with differing toxicities to the larvae (data not shown). The diets were prepared as above. Neonate E. postvittana, as above, were weighed in batches of five and then transferred individually into the AA cups, 20 per treatment, in three blocks each of four treatments. Larvae were maintained at 21 ± 1◦ C. Survival was recorded weekly and larvae were weighed 21 days after ingesting these diets. Pupation time, and weight and sex of pupae were recorded. E. postvittana larvae were considered better candidates for this experiment, where resources were limited, since they are considerably smaller than H. armigera larvae, and consequently ate less diet.
Statistical Analysis During the period of exponential growth of lepidopteran larvae, plots of logarithms of the weights against time form a straight line. Thus larval growth rate over a defined time period during exponential growth is calculated as loge (final weight) − loge (initial weight) (Markwick et al., 1995). Comparisons of mean weights of both larvae and pupae, and mean larval growth rates were analyzed using a one-way analR ysis of variance followed by multiple comparisons using the Tukey method (Minitab version 16).
RESULTS Inhibition by 120 Fruit Extracts Screening of 120 fruit extracts as described in the methods produced a single extract with marked inhibitory effects and five extracts with a lesser inhibitory effect (data not shown). The former was an extract from the peel of apple fruit (M. × domestica cv. “Big Red”), and was selected for further study. This was the solitary peel extract in the collection we screened. Assays of this extract using E. postvittana larvae, confirmed its inhibitory properties against this second lepidopteran species (data not shown). No further studies were conducted on the other five extracts.
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Table 1. Inhibition of lipase activity by successive fractionation of “Big Red” apple peel. An ethanol-soluble fraction of powdered peel was fractionated and assayed as described in the methods. The results are the mean and standard error of two determinations of the same extracts % Control Rate ± SEM
Fraction Unfractionated 10% methanol 30% methanol 50% methanol 70% methanol 100% methanol 100% ethyl acetate 100% dichloromethane
39.0 76.7 94.1 102.1 104.0 18.1 69.7 90.5
± ± ± ± ± ± ± ±
7.5 5.4 7.9 2.1 0.5 0.3 2.9 1.9
Figure 1. The reversed-phase HPLC chromatogram of the components of apple skin that elute from the SPE with 100% methanol fraction (see Table 1). This fraction contained the majority of lipase inhibitory substances found in cv. “Big Red” apple peel (see Tables 1 and 2). The peaks are labeled according to Table 2 viz 6, hydroxyl-ursolic acid; 10, hydroxyl-ursolic acid; 4, oxo-ursolic acid; 9, coumaroyloxy-ursolic acid; 2, oleanolic acid; 8, ursolic acid. The unlabeled peaks are unidentified, lipase noninhibitory compounds.
Fractionation of M. x Domestica cv. “Big Red” Apple Peel Extract The ethanol extract of ‘‘Big Red’’ apple was fractionated by column chromatography and the inhibition of lipase activities for each of the fractions are shown in Table 1. Analysis by HPLC with diode array detection showed that each fraction contained at least 3–6 major peaks and that the profiles (data not shown) were distinct from each other. The results of the lipase assay (Table 1) show that the largest inhibition occurred with the 100% methanol fraction, with constituent solubilities between 70 and 100% MeOH. Analysis and Identification of Lipase Inhibitors in “Big Red” Methanol Fraction The HPLC chromatogram trace of the “Big Red” 100% methanol fraction is presented as Fig. 1 and using preparative HPLC, 12 compounds were isolated in sufficient amounts for use in the lipase inhibition assay; the results are shown in Table 2. LC-MS analysis of the major inhibitory compounds identified them as ursolic acid, oleanolic acid, and hydroxyl-, oxo-, and coumaryl- derivatives of either ursolic or oleanolic acid (Table 2). The relative inhibitory effects could not be established since the amounts in each fraction were not quantified. However an inhibition assay using authentic ursolic acid indicated a LC50 of < 10 μM. Archives of Insect Biochemistry and Physiology
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Table 2. Inhibition by individual compounds in the 100% methanol fraction separated and isolated by preparative HPLC (see Fig. 1). The data are the results of one of three experiments Compound no. 1 2 3 4 5 6 7 8 9 10 11 12
Tentative IDa
% Control rate
– Oleanolic acid – Oxo-ursolic acid – Hydroxyl-ursolic acid – Ursolic acid Coumaroyloxy-ursolic acid Hydroxyl-ursolic acid – –
92.6 56.1 71.5 58.9 87.2 28.5 79.5 17.3 67.3 43.4 77.7 102.7
a
Tentative refers only to the position of the hydroxyl-, oxo- and coumaroyloxy- substituents on ursolic acid being unassigned. Both ursolic acid and oleanolic acid were confirmed using authentic standards.
Table 3. Inhibition of lipase activity by peel extracts of seven apple cultivars. All peel extracts were prepared as DMSO extracts at 10 mg/mL and assayed as described in the methods Cultivar Envy Fuji Pink Lady Big Red Pacific Rose Granny Smith Braeburn
% Control rate 49.3 40.2 29.5 36.4 35.9 29.1 60.9
Inhibition by Apple Peel Extracts from Seven Cultivars To investigate the distribution of triterpene acids in apple, peel extracts were prepared from six additional apple cultivars and compared to inhibition by the M. x domestica cv. “Big Red” peel extract used in the earlier studies (Table 3). All showed moderate inhibition of lipase activity, extracts from cv. “Braeburn” and cv. “Envy” being less inhibitory whereas those from cv. “Granny Smith” and cv. “Pink Lady” were slightly more inhibitory. Effects of Ursolic Acid on Larval Growth and Development We found it difficult to produce an artificial diet containing homogeneously distributed ursolic acid without adding ursolic acid as a solution in DMSO. Therefore, prior to studies on the effect of ursolic acid on E. postvittana larvae, we assessed the effects of DMSO itself on larval growth and development. The initial mean weights of neonate larvae were not significantly different between treatments (F(5,24) = 0.23, P = 0.948) but at 21 days differences between treatments were significant (F(4,78) = 14.49, P < 0.001). There was no significant difference in mean larval weight between larvae fed for 21 days on control diet and diet containing 1% DMSO, but there was a significant difference in weight between larvae fed on control diet and diet containing 2% DMSO (Tukey, P < 0.05). However, amongst those larvae that pupated, there was no significant weight difference Archives of Insect Biochemistry and Physiology
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Table 4. Mean (± SEM) weights (mg) of Epiphyas postvittana larvae as neonates, and after feeding for 21 days on artificial diets containing DMSO and ursolic acid. Growth rates (calculated as loge 21 day weight – loge initial weight) are also shown. (n) = the number of larvae surviving to 21 days from the original 60 neonates transferred onto the treatments Mean larval weight ± SEM (mg) Treatments 1. 2. 3. 4.
No-additive control DMSO control 0.04% ursolic acid 0.16% ursolic acid
Neonate 0.189 0.192 0.180 0.194
± ± ± ±
0.006a 0.005a 0.008a 0.006a
21 days old 19.53 10.73 10.05 8.05
± ± ± ±
1.10a 0.50b 0.66b,c 0.48c
Growth rates ± SEM (n) 4.52 3.96 3.87 3.63
± ± ± ±
0.08 (52)a 0.05 (54)b 0.09 (48)b,c 0.07 (50)c
Means within a column followed by different superscript letters are significantly different (Tukey: P < 0.05).
amongst either male or female pupae on control diets or on diets containing DMSO at 1 or 2%, indicating that the earlier effects of DMSO at these concentrations, on survival and weight, had disappeared by pupation. Because 2% was the lowest concentration of DMSO that would satisfactorily dissolve ursolic acid and because the effects of 2% DMSO were similar to those of 2% ethanol used for our study with tetrahydrolipstatin (Markwick et al., 2011), we used 2% DMSO in subsequent experiments. There were no significant differences in mean (± SEM) neonate weight (mg) between the three blocks (replicates) within each treatment (P > 0.5) or between the four treatments (F3,47 0.91, P = 0.444) (Table 4). However, after larvae had fed for 21 days on control and treatment diets containing DMSO with or without ursolic acid, there were significant differences in mean larval weight between treatments (although not between blocks within each treatment, that is, no replicate effect). The difference in mean larval weight between the control with no additives and the control with 2% DMSO added was significant (Tukey, P < 0.05), that is, the DMSO additive had a significant effect on the growth of the larvae (Table 4). Mean larval weight at 21 days on the diet containing 0.04% ursolic acid was lower than that on diet containing the DMSO solvent alone but this was not statistically significant (Tukey, P > 0.05). However, both the mean weight and growth rate of larvae fed for 21 days on diet containing 0.16% ursolic acid were significantly lower than those for both the no-additive and DMSO control treatments (Table 4). Thus ursolic acid at 0.16% has a significant effect on the growth rate of E. postvittana larvae over the first 21 days, over and above the negative effect of 2% DMSO on larval growth. There was little effect on survival however; note (n) the number of survivors in each treatment to 21 days (Table 4). At pupation, because of the sexual dimorphism in size, male and female pupal weights were analyzed separately. Amongst males, weight differences between the treatments were significant (ANOVA: F3,92 = 10.97, P < 0.001). However, when the no-additive control treatment was removed, there was no significant difference in mean pupal weights between the DMSO control and the two ursolic acid treatments (Table 5). Amongst females, ANOVA also indicated a significant difference (F3, 83 = 4.44, P = 0.06) between treatments, but as with the males, mean female pupal weights were not significantly different between the ursolic acid treatments and the DMSO control. The mean female pupal weight was significantly higher in the no-additive control than in the DMSO control and the 0.16% ursolic acid treatment, but not significantly different from the 0.04% ursolic acid treatment (Table 5). Archives of Insect Biochemistry and Physiology
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Table 5. Mean (± SEM) weights (mg) at pupation, and duration of development to pupation (mean ± SEM days) for Epiphyas postvittana larvae fed since neonates on artificial diets containing DMSO and ursolic acid. (n) = the number of larvae surviving to pupation from the original 60 neonates transferred onto the treatments Mean pupal weight ± SEM (mg) Treatments 1. 2. 3. 4.
water control DMSO control 0.04% ursolic acid 0.16% ursolic acid
Male (n) 31.86 27.58 27.95 27.88
± ± ± ±
0.44 (25)a 0.57 (24)b 0.83 (22)b 0.64 (25)b
Female (n) 50.42 48.59 53.49 46.59
± ± ± ±
1.48 (19)ab 1.72 (19)b 1.28 (24)a 1.41 (25)b
Mean days ± SEM to pupation Male 31.40 34.42 33.68 35.42
± ± ± ±
0.48a 0.59b 0.47b 0.66b
Female 35.20 38.77 38.74 41.72
± ± ± ±
0.63a 1.25b 0.98ab 0.91b
Means within a column followed by different superscript letters are significantly different (Tukeys: P < 0.05).
Ursolic acid treatments also increased the time to pupation; the mean number of days to pupation for each treatment is given in Table 5. The mean time to pupation for both males and females was significantly longer in two treatments (DMSO and 0.16% ursolic acid) than in the no-additive control, but there was no significant difference between the DMSO and ursolic acid treatments. These treatments generally showed higher variability in pupal weights.
DISCUSSION The activity-based purification of the active lipase-inhibitory components of a methanolic apple peel extract were found to be triterpene acids; ursolic acid, oleanolic acid, and monohydroxy-, keto- and coumaroyloxy-dervatives of these compounds. The presence of ursolic and oleanolic acids in apple skin (Belding et al., 1998, Frighetto et al., 2008, Andre et al., 2012) is well known and has been previously reported in apple leaves too (Bringe et al., 2006). In both tissues they are contained within the epicuticular wax and form a major component thereof. Our finding (Table 3) that cv. “Fuji” extracts contains higher inhibitory activity than cv. “Granny Smith” is consistent with those of Frighetto et al. (2008) that ursolic acid was higher in cv. “Fuji” than in cv. “Granny Smith.” The data of Andre et al. (2012) indicate dramatic variation, between 0.004 and 0.35% of apple skin is ursolic acid and between 0.004 and 0.08% is oleanolic acid, depending on cultivar. Several authors have reported the effect of ursolic acid and related compounds on PL activity. Jang et al. (2008) reported five triterpene acids (including ursolic acid) from the roots of Actinidia arguta inhibited PL and Kim et al. (2009) reported that ursolic acid from the same source inhibited phosphodiesterase activity. Ninomiya et al. (2004) reported inhibition of PL from Salvia officinalis leaves and inhibition of PL by triterpene glycosides and saponins have also been noted (Morikawa and Yoshikawa, 2004; Xu et al., 2005; Yoshizumi et al., 2006). The mode of inhibition of these compounds is largely unclarified since normal solution kinetics are not applicable to heterogeneous lipase assays. Our data are consistent with these studies but report inhibition from a completely different enzyme source. While earlier studies have focused on inhibition of PL with an interest on dietary supplementation using ursolic acid as an antiobesity factor, our work has used an enzyme preparation that contains galactolipase: TAG lipase activities in a 2.6:1 ratio (Christeller et al., 2011). The extent of inhibition observed indicates that both Archives of Insect Biochemistry and Physiology
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activities are inhibited. Genomic studies (Simpson et al., 2007; Christeller, unpublished data) further show that while lepidopteran midgut galactolipases are members of the pancreatic lipase superfamily, the TAG lipases are members of the gastric lipase superfamily. To our knowledge, no studies showing effects of these compounds on vertebrate gastric lipases have been reported but our results suggest that they are likely to be inhibited. Insect midgut galactolipases and TAG lipases are inhibited not only by ursolic acid but also by oleanolic acid and by some triterpene acid derivatives (Table 2). While we have previously shown that lepidopteran larval midgut lipase activities are sensitive to the lipase inhibitor, tetrahydrolipstatin (Markwick et al., 2011), this is the first report that these lipase activities are inhibited by plant-derived compounds. Our findings (Table 3) that variation in lipase inhibitory activity occurs between cultivars and is consistent with being associated with the triterpene acid components together indicate that the potential to enhance triterpene acid levels in apple skin of elite cultivars exists, either by direct measurement or by determining genetic markers for triterpene acid accumulation. The function(s) of triterpene acids in plants is unclear. They accumulate in diverse forms in the intracuticlular layer (Buschhaus and Jetter, 2011) of many, perhaps most, even all plants. Their diversity is surely attributable to the broad specificity of plant P450s for which they are substrates and their properties may be important to the chemical properties of functioning cuticles (Vogg et al., 2004; Yu et al., 2007). Whether their presence evolved to contribute to pest deterrence or fungal resistance, with many fungi including cutinase and other lipases (Reis et al., 2005; Voigt et al., 2005; Bravo-Ruiz et al., 2013) among their pathological determinants, awaits further studies. Our preliminary bioassay showed that DMSO at a concentration at 2% in diets had a significant effect on the growth and development of E. postvittana larvae. Taking this into account, there was a further significant effect of ursolic acid on larval growth and development in this species: at 0.16%, ursolic acid significantly reduced the larval weight and growth rate during the first 21 day feeding. However, at pupation, mean weights were not significantly different in DMSO treatments with and without ursolic acid, although in all three treatments, mean pupal weights were significantly lower than those in the noadditive control. Time to pupation was delayed in the ursolic acid treatments, although this was also not significantly different from the DMSO control. Survival was not significantly affected by ingestion of ursolic acid at concentrations used in this experiment. Therefore, the main impact of ursolic acid treatments was early in the life-cycle/development of the E. postvittana larvae. It seems likely that neonates are deficient in long-chain polyunsaturated fatty acids but accumulate adequate amounts under the experimental conditions within 3 weeks to restore normal growth rates. The values of 0.04 and 0.16% ursolic acid in diet used in our experiment are at the mid to lower end of ursolic acid values reported in apple skin (Andre et al., 2012)with only two russet varieties of 109 apple varieties measured with values below 0.02% and where up to 2.2-fold higher values (0.35%) were found. Our data, reporting a significant effect on initial growth rates, suggest that ursolic acid might act as a growth inhibitor in some cultivars with high concentration but not in others with low concentrations. Bioassays on intact apple fruit from a range of cultivars may support this conclusion. The behavior of the larvae of E. postvittana might also indicate a role for ursolic acid effects. Eggs are laid on leaves and neonate larvae prefer to feed on shoot tips (Geier and Briese, 1980; Lo et al., 2000). Other leaves or leaf pairs are utilized as the preferred sites are occupied, and feeding on fruit (Geier and Briese, 1980; Lo et al., 2000) or more commonly leaf-fruit pairs, is only observed later in the season when the fruit area is proportionally higher Archives of Insect Biochemistry and Physiology
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(Suckling and Ioratti, 1996). Bringe et al. (2006) noted that the concentration of ursolic acid is lower in leaves and declines rapidly as leaves age. It is probable that larvae feed on fruit only by chance as other feeding sites become scarcer; thus higher concentrations of ursolic acid might deter even this limited feeding activity. Fruit-feeding lepidopteran larvae such as those of Cydia pomonella seem to avoid feeding on the skin by burrowing directly into the center of the fruit. Increasing ursolic acid may also improve fruit health attributes with a plethora of in vitro, cell-based and mouse model studies reported in the literature (e.g., Kang et al., 2008; Jang et al., 2009; Wang et al., 2010; Jang et al., 2010; Kunkel et al., 2011; Rao et al., 2011). Health benefits of triterpenoids have been recently reviewed (Szakiel et al., 2012) but would be, however, contingent on consumers eating the skin of their apples. A breeding program, either by direct analysis or by marker-assisted breeding, for increased triterpene acids in the peel of apple and potentially other fruits, could therefore meet dual objectives of insect feeding deterrence and improved health properties for consumers.
ACKNOWLEDGMENTS We would like to thank Dr. A. Barrington for providing the insects and Dr. D. Rowan and Dr. W. Laing for useful discussions. Neither conflicts of interest, financial or otherwise, nor any breaches of ethical standards were identified by the authors.
Literature Cited Andre CM, Greenwood JM, Walker EG, Rassam M, Sullivan M, Evers D, Perry NB, Laing WA. 2012. Anti-inflammatory procyanidins and triterpenes in 109 apple varieties. J Agriculture Food Chem 60: 10546–10554. Belding RD, Blankenship SM, Young E, Leidy RB. 1998, Composition and variability of epicuticular waxes in apple cultivars. J Amer Soc Hort Sci 123: 348–356. Bravo-Ruiz G, Ruiz-Roldan C, Roncero MIG. 2013. Lipolytic System of the tomato pathogen Fusarium oxysporum f. sp lycopersici. Molec Plant-Microbe Interactions 26: 1054–1067. Bringe K, Schumacher CFA, Schmitz-Eiberger M, Steiner U, Oerke EC. 2006. Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces. Phytochem 67: 161–170. Buschhaus C, Jetter R. 2011. Composition differences between epicuticular and intracuticular wax substructures: How do plants seal their epidermal surfaces? J Exp Bot 62: 841–853. Canavoso LE, Jouni ZE, Karnas KJ, Pennington JE, Wells MA. 2001. Fat metabolism in insects. Ann Review Nutrition 21: 23–46. Christeller JT, Amara S, Carriere F. 2011. Galactolipase, phospholipase and triacylglycerol lipase activities in the midgut of six species of lepidopteran larvae feeding on different lipid diets. J Insect Physiol 57: 1232–1239. Christeller JT, Poulton J, Markwick NM, Simpson RM. 2010. The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth Epiphyas postvittana Walker Tortricidae. Insect Molec Biol 191: 9–25. Dadd RH. 1985. Nutrition, organisms. In: Kerkut GA, Gilbert LI, editors. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Oxford, UK: Pergamon Press. p. 313–390. Delorme V, Dhouib R, Canaan S, Fotiadu F, Carriere F, Cavalier JF. 2011. effects of surfactants on lipase structure, activity, and inhibition. Pharm Res 8: 1831–1842. Archives of Insect Biochemistry and Physiology
148
r
Archives of Insect Biochemistry and Physiology, July 2014
Frighetto RTS, Welendorf RM, Nigro EN, Frighetto N, Siani AC. 2008. Isolation of ursolic acid from apple peels by high speed counter-current chromatograph. Food Chem 106: 767–771. Gargouri Y, Chahinian H, Moreau, H, Ransac S, Verger R. 1991. Inactivation of Pancreatic and Gastric Lipases by THL and C12-o-TNB: A kinetic study with emulsified tributyrin. Biochim Biophys Acta 1085: 322–328. Geier PW, Briese DT. 1980. The light-brown apple moth, Epiphyas postvittana (Walker): 4. Studies on the population dynamics and injuriousness to apples in the Australian Capital Territory. Aust J Ecol 5: 63–93. Han LK, Kimura Y, Kawashima M, Takaku T, Taniyama T, Hayashi T, Zheng YN, Okuda H. 2001. Anti-obesity effects in rodents of dietary teasaponin, a lipase inhibitor. Internat J Obesity 25: 1459–1464. Hatano T, Yamashita A, Hashimoto T, Ito H, Kubo N, Yoshiyama M, Shimura S, Itoh Y, Okuda T, Yoshida T. 1997. Flavan dimers with lipase inhibitory activity from Cassia nomame. Phytochem 46: 893–900. Jang DS, Lee GY, Kim J, Lee YM, Kim JM, Kim YS, Kim JS. 2008. A new pancreatic lipase inhibitor isolated from the roots of Actinidia arguta. Arch Pharmacal Res 31: 666–670. Jang S-M, Yee S-T, Choi J, Choi M-S, Do G-M, Jeon S-M, Yeo J, Kim M-J, Seo K-I, Lee M-K. 2009. Ursolic acid enhances the cellular immune system and pancreatic beta-cell function in streptozotocininduced diabetic mice fed a high-fat diet. Internat Immunopharmacol 9: 113–119. Jang S-M, Kim M-J, Choi M-S, Kwon E-Y, Lee M-K. 2010. Inhibitory effects of ursolic acid on hepatic polyol pathway and glucose production in streptozotocin-induced diabetic mice. Clinical Exp Metabol 59: 512–519. Kang SY, Yoon SY, Roh DH, Jeon MJ, Seo HS, Uh DK, Kwon YB, Kim HW, Han HJ, Lee HJ, Lee JH. 2008. The anti-arthritic effect of ursolic acid on zymosan-induced acute inflammation-induced chronic arthritis models and adjuvant. J Pharmacy Pharmacol 60: 1347–1354. Karu N, Reifen R, Kerem Z. 2007. Weight gain reduction in mice fed Panax ginseng saponin, a pancreatic lipase inhibitor. J Agricultural Food Chem 55: 2824–2828. Kim J, Jang DS, Kim H, Kim JS. 2009. Anti-lipase and lipolytic activities of ursolic acid isolated from the roots of Actinidia arguta. Arch Pharmacal Res 32: 983–987. Kunkel SD, Suneja M, Ebert SM, Bongers KS, Fox DK, Malmberg SE, Alipour F, Shields RK, Adams CM. 2011. mRNA Expression Signatures of Human Skeletal Muscle Atrophy Identify a Natural Compound that Increases Muscle Mass. Cell Metabol 13: 627–638. Lo PL, Suckling DM, Bradley SJ, Walker JTS, Shaw PW, Burnip GM. 2000. Factors affecting feeding site preferences of lightbrown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae) on apple trees in New Zealand. New Zealand J Crop Food Sci 28: 235–243. Markwick NP, Poulton J, McGhie TK, Wohlers MW, Christeller JT. 2011. The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana Walker Tortricidae, Lepidoptera. J Insect Physiol 5712: 1643–1650. Markwick NP, Laing WA, Christeller JT, McHenry JZ, Newton MR. 1998. Overproduction of digestive enzymes compensates for inhibitory effects of protease and alpha-amylase inhibitors fed to three species of leafrollers Lepidoptera, Tortricidae. J Economic Entomol 916: 1265–1276. Markwick NP, Reid SJ, Laing WA, Christeller JT. 1995. Effects of dietary protein and protease inhibitors on codling moth (Lepidoptera: Tortricidae). J Economic Entomol 88: 33–39. McGhie T K, Hudault S, Lunken RCM, Christeller JT. 2012. Apple peels, from seven cultivars, have lipase-inhibitory activity and contain numerous ursenoic acids as identified by LC-ESI-QTOFHRMS. J Agricultural Food Chem 601: 482–491. McManus MT, Burgess EPJ. 1995. Effects of the soybean kunitz trypsin-inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 41: 731–738.
Archives of Insect Biochemistry and Physiology
Triterpene Acids Inhibit Larval Growth
r
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Morikawa T, Xie YY, Asao Y, Okamoto M, Yamashita C, Muraoka O, Matsuda H, Pongpiriyadacha Y, Yuan D, Yoshikawa M. 2009. Oleanane-type triterpene oligoglycosides with pancreatic lipase inhibitory activity from the pericarps of Sapindus rarak. Phytochem 70: 1166–1172. Nilsson-Ehle P, Schotz MC. 1976. A stable, radioactive substrate emulsion for assay of Iipoprotein Iipase. J Lipid Res 17: 536–541. Ninomiya K, Matsuda H, Shimoda H, Norihisa N, Kasajima N, Yoshino T, Morikawa T, Yoshikawa M. 2004. Carnosic acid, a new class of lipid absorption inhibitor from sage. Bioorg Med Chem Lett 14: 1943–1946. Rao VS, de Melo CL, Queiroz MGR, Lemos TLG, Menezes DB, Melo TS, Santos FA. 2011. Ursolic acid, a pentacyclic triterpene from Sambucus australis, prevents abdominal adiposity in mice fed a high-fat diet. J Med Food 14: 1375–82. Reis H, Pfiffi S, Hahn M. 2005. Molecular and functional characterization of a secreted lipase from Botrytis cinerea. Mol Plant Pathol 6: 257–267. Satouchi K, Hirano K, Fujino O, Ikoma M, Tanaka T, Kitamura K. 1998. Lipoxygenase-1 from soybean seed inhibiting the activity of pancreatic lipase. Biosci Biotechnol Biochem 62: 1498– 1503. Satouchi K, Kodama Y, Murakami K, Tanaka T, Iwamoto H, Ishimoto M. 2002. A lipaseinhibiting protein from lipoxygenase-deficient soybean seeds. Biosci Biotechnol Biochem 66: 2154–2160. Sheng L, Qian ZY, Zheng SG, Xi L. 2006. Mechanism of hypolipidemic effect of crocin in rats, Crocin inhibits pancreatic lipase. Europ J Pharmacol 543: 116–122. Shin JE, Han MJ, Kim DH. 2003. 3-Methylethergalangin isolated from Alpinia officinarum inhibits pancreatic lipase. Biol Pharm Bul 26: 854–857. Shin JE, Han MJ, Song MC, Baek NI, Kim DH. 2004. 5-hydroxy-7–4 ‘-hydroxy-3 ‘-methoxyphenyl1-phenyl-3-heptanone: A pancreatic lipase inhibitor isolated from Alpinia officinarum. Biol Pharm Bul 27: 138–140. Simpson RM, Newcomb RD, Gatehouse HS, Crowhurst RN, Chagne D, Gatehouse LN, Markwick NP, Beuning LL, Murray C, Marshall SD, Yauk YK, Nain B, Wang YY, Gleave AP, Christeller JT. 2007. Expressed sequence tags from the midgut of Epiphyas postvittana Walker Lepidoptera, Tortricidae. Insect Molec Biol 16: 675–690. Slansky FJ, Scriber JM. 1985. Food Consumption and Utilization. In: Kerkut GA, Gilbert LI, editors. Comprehensive Insect Physiology Biochemistry and Pharmacology. Oxford: Pergamon Press. p. 87–163. Suckling DM, Ioratti C. 1996. Behavioral responses of leafroller larvae to apple leaves and fruit. Entomol Exp Appl 81: 97–103. Suckling DM, Markwick NP, Wigley PJ, Frater CM, Chilcott CN, Maindonald JH. 1996. Response to purified Cry 1B protein in lab-wild type crosses of Light Brown Apple Moth. Proceedings of 49th New Zealand Plant Protection Conference 49: 64–70. Szakiel A, Paczkowski C, Pensec F, Bertsch C. 2012. Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochem Rev 11: 263–284. Voigt CA, Schafer W, Salomon S. 2005. A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals. Plant J 42: 364–375. Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M. 2004. Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase. J Exp Bot 55: 1401– 1410. Wang ZH, Hsu CC, Huang CN, Yin MC. 2010. Anti-glycative effects of oleanolic acid and ursolic acid in kidney of diabetic mice. Europ J Pharmacol 628: 255–260. Won SR, Kim SK, Kim YM, Lee PH, Ryu JH, Kim JW, Rhee HI. 2007. Licochalcone A, A lipase inhibitor from the roots of Glycyrrhiza uralensis. Food Res Internat 40: 1046–1050. Archives of Insect Biochemistry and Physiology
150
r
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Xie WD, Zhang Y, Wang NL, Zhou H, Du LJ, Ma XH, Shi XJ, Cai GP. 2008. Novel effects of macrostemonoside A, a compound from Allium macrostemon Bung, on hyperglycemia, hyperlipidemia, and visceral obesity in high-fat diet-fed C57BL/6 mice. Europ J Pharmacol 599: 159–165. Xu BJ, Han LK, Zheng YN, Lee JH, Sung CK. 2005. In vitro inhibitory effect of triterpenoidal saponins from Platycodi Radix on pancreatic lipase. Arch Pharmacal Res 28: 180–185. Yamada K, Murata T, Kobayashi K, Miyase T, Yoshizaki F. 2010. A lipase inhibitor monoterpene and monoterpene glycosides from Monarda punctata. Phytochem 71: 1884–1891. Yamamoto M, Shimura S, Itoh Y, Ohsaka T, Egawa M, Inoue S. 2000. Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs, Nomame herba, on rats fed a high-fat diet. Internat J Obesity 24: 758–764. Yoshizumi K, Hirano K, Ando H, Hirai Y, Ida Y, Tsuji T, Tanaka T, Satouchi K, Terao, J. 2006. Lupane-type saponins from leaves of Acanthopanax sessiliflorus and their inhibitory activity on pancreatic lipase. J Agricultural Food Chem 54: 335–341. Yu MML, Schulze HG, Jetter R, Blades MW, Turner RFB. 2007. Raman microspectroscopic analysis of triterpenoids found in plant cuticles. Appl Spectrosc 61: 32–37.
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