Formation Of Glucosides Research Articles

Methyl jasmonate induced tolerance effect of Pinus koraiensis to Bursaphelenchus xylophilus.

Methyl jasmonate (MeJA) can affect the balance of hormones and regulate the disease resistance of plants. Exploring the application and mechanism of MeJA in inducing the tolerance of Pinus koraiensis to pine wood nematode (PWN) infection is of great significance for developing new strategies for pine wilt disease control. Different concentrations (0.1, 1, 5 and 10 mm) of MeJA treatment groups showed differences in relative tolerance index and relative anti-nematode index of P. koraiensis seedlings to PWN infection. The treatment of 5 mm MeJA solution induced the best tolerance effect, followed by the 1 mm MeJA solution. Transcriptome analysis indicated that many plant defense-related genes upregulated after treatment with 1, 5 and 10 mm MeJA solutions. Among them, genes such as jasmonate ZIM domain-containing protein, phenylalanine ammonia-lyase and peroxidase also continuously upregulated after PWN infection. Metabolome analysis indicated that jasmonic acid (JA) was significantly increased at 7 days postinoculation with PWN, and after treatment with both 1 and 5 mm MeJA solutions. Integrated analysis of transcriptome and metabolome indicated that differences in JA accumulation might lead to ubiquitin-mediated proteolysis, and expression changes in trans-caffeic acid and trans-cinnamic acid-related genes, leading to the abundance differences of these two metabolisms and the formation of multiple lignin and glucosides. MeJA treatment could activate the expression of defense-related genes that correlated with JA, regulate the abundance of defense-related secondary metabolites, and improve the tolerance of P. koraiensis seedlings to PWN infection. © 2024 Society of Chemical Industry.

Heterologous expression of PtAAS1 reveals the metabolic potential of the common plant metabolite phenylacetaldehyde for auxin synthesis in planta.

Aromatic aldehydes and amines are common plant metabolites involved in several specialized metabolite biosynthesis pathways. Recently, we showed that the aromatic aldehyde synthase PtAAS1 and the aromatic amino acid decarboxylase PtAADC1 contribute to the herbivory-induced formation of volatile 2-phenylethanol and its glucoside 2-phenylethyl-β-D-glucopyranoside in Populus trichocarpa. To unravel alternative metabolic fates of phenylacetaldehyde and 2-phenylethylamine beyond alcohol and alcohol glucoside formation, we heterologously expressed PtAAS1 and PtAADC1 in Nicotiana benthamiana and analyzed plant extracts using untargeted LC-qTOF-MS and targeted LC-MS/MS analysis. While the metabolomes of PtAADC1-expressing plants did not significantly differ from those of control plants, expression of PtAAS1 resulted in the accumulation of phenylacetic acid (PAA) and PAA-amino acid conjugates, identified as PAA-aspartate and PAA-glutamate. Herbivory-damaged poplar leaves revealed significantly induced accumulation of PAA-Asp, while levels of PAA remained unaltered upon herbivory. Transcriptome analysis showed that members of auxin-amido synthetase GH3 genes involved in the conjugation of auxins with amino acids were significantly upregulated upon herbivory in P. trichocarpa leaves. Overall, our data indicates that phenylacetaldehyde generated by poplar PtAAS1 serves as a hub metabolite linking the biosynthesis of volatile, non-volatile herbivory-induced specialized metabolites, and phytohormones, suggesting that plant growth and defense can be balanced on a metabolic level.

Linker length-dependent morphologies in self-assembled structures of anthracene glucosides

Anthracenemethyl glucosides, that possess ethylene glycol linkers connecting the glucoside with anthracene moiety, are studied herein. Koenigs-Knorr glycosylation of ethylene glycol-tethered anthracene with acetobromo glucose, followed by removal of the protecting groups, lead to the facile formation of the target glucosides. Aq. solutions of these anthracene glucosides readily undergo self-assembly, with critical aggregation concentration varying between 0.4 and 1 mM, depending on the linker, being ethylene-, di- and tetraethylene glycol, as assessed by photophysical evaluations. Circular dichroism spectra show chiral self-assembled structures for these glucosides in solution, from which a left-handed chirality is adjudged. Morphologies of the self-assembled structures of these glucosides are controlled by the linker length. With the ethylene glycol linker, vesicles form initially, around which tendrils start to grow as the concentration of the glucoside is increased. Whereas, di- and tetraethylene glycol-spaced glucosides prefer agglomerated fractal-like structures, as assessed by microscopies. The aggregation phenomenon in the latter glucosides appears to be under the non-equilibrium-driven, dissipative control.

Biosynthesis, herbivore induction, and defensive role of phenylacetaldoxime glucoside.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

Multi-omics reveal key enzymes involved in the formation of phenylpropanoid glucosides in Artemisia annua

Although mainly known for producing artemisinin, Artemisia annua is enriched in phenylpropanoid glucosides (PGs) with significant bioactivities. However, the biosynthesis of A. annua PGs is insufficiently investigated. Different A. annua ecotypes from distinct growing environments accumulate varying amounts of metabolites, including artemisinin and PGs such as scopolin. UDP-glucose:phenylpropanoid glucosyltransferases (UGTs) transfers glucose from UDP-glucose in PG biosynthesis. Here, we found that the low-artemisinin ecotype GS produces a higher amount of scopolin, compared to the high-artemisinin ecotype HN. By combining transcriptome and proteome analyses, we selected 28 candidate AaUGTs from 177 annotated AaUGTs. Using AlphaFold structural prediction and molecular docking, we determined the binding affinities of 16 AaUGTs. Seven of the AaUGTs enzymatically glycosylated phenylpropanoids. AaUGT25 converted scopoletin to scopolin and esculetin to esculin. The lack of accumulation of esculin in the leaf and the high catalytic efficiency of AaUGT25 on esculetin suggest that esculetin is methylated to scopoletin, the precursor of scopolin. We also discovered that AaOMT1, a previously uncharacterized O-methyltransferase, converts esculetin to scopoletin, suggesting an alternative route for producing scopoletin, which contributes to the high-level accumulation of scopolin in A. annua leaves. AaUGT1 and AaUGT25 responded to induction of stress-related phytohormones, implying the involvement of PGs in stress responses.

Leaf Lipid Alterations in Response to Heat Stress of Arabidopsis thaliana.

In response to elevated temperatures, plants alter the activities of enzymes that affect lipid composition. While it has long been known that plant leaf membrane lipids become less unsaturated in response to heat, other changes, including polygalactosylation of galactolipids, head group acylation of galactolipids, increases in phosphatidic acid and triacylglycerols, and formation of sterol glucosides and acyl sterol glucosides, have been observed more recently. In this work, by measuring lipid levels with mass spectrometry, we confirm the previously observed changes in Arabidopsis thaliana leaf lipids under three heat stress regimens. Additionally, in response to heat, increased oxidation of the fatty acyl chains of leaf galactolipids, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols, and incorporation of oxidized acyl chains into acylated monogalactosyldiacylglycerols are shown. We also observed increased levels of digalactosylmonoacylglycerols and monogalactosylmonoacylglycerols. The hypothesis that a defect in sterol glycosylation would adversely affect regrowth of plants after a severe heat stress regimen was tested, but differences between wild-type and sterol glycosylation-defective plants were not detected.

Synthesis of butyl glucoside over sulphated Zr-SBA-15 and tungstophosphoric acid incorporated SBA-15 catalysts

Glycosidation of glucose with n-butanol was studied over sulphated Zr incorporated SBA-15 and tungstophosphoric acid (TPA) incorporated SBA-15 catalysts. The catalysts were prepared with different Zr, TPA and SO4 amounts via hydrothermal synthesis. SBA-15 structure was preserved and Zr and TPA were successfully incorporated. Sulphation improved the acidity of Zr-SBA-15 and the ratio of Brønsted to Lewis acid sites (B/L) increased. Thus, much higher butyl glucoside yields (about 70 %) were obtained over SO4/Zr-SBA-15 catalysts. However, 15 % activity loss was observed after 2 re-use due to sulphate leaching. TPA-SBA-15 catalysts had the highest B/L and provided very high catalytic activity with butyl glucoside yields above 95 %. The initial rate of butyl glucoside formation over TPA-SBA-15 catalysts were close to that of H2SO4. These catalysts were found stable and reusable. Reaction temperature and catalysts amount was studied as the reaction parameters over the most active and selective catalyst.

Aqueous and alcoholic adducts of steviol and steviol glycosides in food products containing stevia

High content of steviol glycosides in stevia leaves is a cause of their high popularity as.a natural sweetener of various sugar-free food products. Stevioside (13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester) is one of the main steviol glycosides in stevia leaves known for its hydrolytic instability responsible for the formation of simple steviol glucosides (steviolbioside, rubusoside, steviol monoside) and steviol. However, the formation of hydroxy and alkoxy adducts of stevioside and of its hydrolysis products has not yet been reported. The performed experiments prove that water and alkoxy adducts are formed not only during temperature processing of stevioside but also of stevia and stevia-containing food products. Their quantities depend on environment pH, water concentration and food composition. Although they are formed in small amounts their biological activity is unknown and should be recognized.

Phenolic composition of rooibos changes during simulated fermentation: Effect of endogenous enzymes and fermentation temperature on reaction kinetics.

Phenolic compounds of Aspalathus linearis (rooibos) are susceptible to oxidation during "fermentation", a process characterized by the formation of a red-brown leaf color. The role of enzymes in this process is not yet understood. An experiment with dried green rooibos plant material pre-treated at 170 °C for 30 min to denature and "inactivate" endogenous enzymes was conducted to confirm the role of oxidative enzymes. The phenolic composition of "enzyme inactivated" plant material was not significantly (p ≥ .05) affected by simulated fermentation, compared to control samples, as determined using piece-wise multivariate analysis of variance for successive time intervals. This proves that rooibos enzymes participate in the oxidation of phenolic compounds during fermentation of the plant material. A kinetic modeling approach was subsequently used to establish reaction kinetic parameters for selected rooibos phenolic compounds. The degradation of aspalathin and nothofagin and formation of eriodictyol glucosides during simulated fermentation at four temperatures from 37 to 50 °C were best described by the fractional conversion model based on first-order kinetics (r2 > 0.98), which allows for non-zero equilibrium concentrations. The extent of degradation for other compounds was too low to enable kinetic modeling. Reaction rates for the degradation/formation of phenolic compounds during fermentation followed the Arrhenius law. Less phenolic degradation (higher equilibrium concentration), but a higher reaction rate constant, was observed at higher temperatures, which could possibly be attributed to inactivation of enzymes.

Regioselective O-glycosylation of flavonoids by fungi Beauveria bassiana, Absidia coerulea and Absidia glauca

In the present study, the species: Beauveria bassiana, Absidia coerulea and Absidia glauca were used in biotransformation of flavones (chrysin, apigenin, luteolin, diosmetin) and flavanones (pinocembrin, naringenin, eriodictyol, hesperetin). The Beauveria bassiana AM 278 strain catalyzed the methylglucose attachment reactions to the flavonoid molecule at positions C7 and C3′. The application of the Absidia genus (A. coerulea AM 93, A. glauca AM 177) as the biocatalyst resulted in the formation of glucosides with a sugar molecule present at C7 and C3′ positions of flavonoids skeleton. Nine of obtained products have not been previously reported in the literature.

214 - Inhibition of Resveratrol Glucosides against AGEs Formation

Resveratrol and its derivatives are the most important sources of dietary stilbenoids. Resveratrol’s glucosides such as resveratrol 3- O-β -D-glucoside and resveratrol 2- O-β -D-glucoside possess many advantages than resveratrol, such as higher hydrophilic and apparent benefits. Herein, the inhibition against fructose-derived advanced glycation end products (AGEs) formation of resveratrol glucosides was compared. The position of glucoside was found to be an important factor affecting inhibitory effect against AGEs formation. Resveratrol O -glucosides attached in the position of C-2, C-3, C-3' or C-4' showed slightly higher inhibition against AGEs formation than that of resveratrol. The anti-AGEs activity is correlated with DPPH radical scavenging capacity but not the methylglyoxal trapping capacity.

Vanillin biosynthetic pathways in plants.

The present review compiles the up-to-date knowledge on vanillin biosynthesis in plant systems to focus principally on the enzymatic reactions of in planta vanillin biosynthetic pathway and to find out its impact and prospect in future research in this field. Vanillin, a very popular flavouring compound, is widely used throughout the world. The principal natural resource of vanillin is the cured vanilla pods. Due to the high demand of vanillin as a flavouring agent, it is necessary to explore its biosynthetic enzymes and genes, so that improvement in its commercial production can be achieved through metabolic engineering. In spite of significant advancement in elucidating vanillin biosynthetic pathway in the last two decades, no conclusive demonstration had been reported yet for plant system. Several biosynthetic enzymes have been worked upon but divergences in published reports, particularly in characterizing the crucial biochemical steps of vanillin biosynthesis, such as side-chain shortening, methylation, and glucoside formation and have created a space for discussion. Recently, published reviews on vanillin biosynthesis have focused mainly on the biotechnological approaches and bioconversion in microbial systems. This review, however, aims to compile in brief the overall vanillin biosynthetic route and present a comparative as well as comprehensive description of enzymes involved in the pathway in Vanilla planifolia and other plants. Special emphasis has been given on the key enzymatic biochemical reactions that have been investigated extensively. Finally, the present standpoint and future prospects have been highlighted.

Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes.

Use of the immunosuppressant mycophenolic acid (MPA) in cats is limited because MPA elimination depends on glucuronidation, which is deficient in cats. We evaluated formation of major (phenol glucuronide) and minor (acyl glucuronide, phenol glucoside, and acyl glucoside) MPA metabolites using liver microsomes from 16 cats, 26 dogs, and 48 humans. All MPA metabolites were formed by human liver microsomes, while dog and cat liver microsomes formed both MPA glucuronides, but only one MPA glucoside (phenol glucoside). Intrinsic clearance (CLint) of MPA for phenol glucuronidation by cat liver microsomes was only 15-17% that of dog and human liver microsomes. However, CLint for acyl glucuronide and phenol glucoside formation in cat liver microsomes was similar to or greater than that for dog and human liver microsomes. While total MPA conjugation CLint was generally similar for cat liver microsomes compared with dog and human liver microsomes, relative contributions of each pathway varied between species with phenol glucuronidation predominating in dog and human liver microsomes and phenol glucosidation predominating in cat liver microsomes. MPA conjugation variation between cat liver microsomes was threefold for total conjugation and for phenol glucosidation, sixfold for phenol glucuronidation, and 11-fold for acyl glucuronidation. Our results indicate that total MPA conjugation is quantitatively similar between liver microsomes from cats, dogs, and humans despite large differences in the conjugation pathways that are utilized by these species.

Two UGT84 Family Glycosyltransferases Catalyze a Critical Reaction of Hydrolyzable Tannin Biosynthesis in Pomegranate (Punica granatum).

Hydrolyzable tannins (HTs) play important roles in plant herbivore deterrence and promotion of human health. A critical step in HT production is the formation of 1-O-galloyl-β-D-glucopyranoside (β-glucogallin, ester-linked gallic acid and glucose) by a UDP-glucosyltransferase (UGT) activity. We cloned and biochemically characterized four candidate UGTs from pomegranate (Punica granatum), of which only UGT84A23 and UGT84A24 exhibited β-glucogallin forming activities in enzyme assays. Although overexpression and single RNAi knockdown pomegranate hairy root lines of UGT84A23 or UGT84A24 did not lead to obvious alterations in punicalagin (the prevalent HT in pomegranate) accumulation, double knockdown lines of the two UGTs resulted in largely reduced levels of punicalagins and bis-hexahydroxydiphenyl glucose isomers. An unexpected accumulation of galloyl glucosides (ether-linked gallic acid and glucose) was also detected in the double knockdown lines, suggesting that gallic acid was utilized by an unidentified UGT activity for glucoside formation. Transient expression in Nicotiana benthamiana leaves and immunogold labeling in roots of pomegranate seedlings collectively indicated cytosolic localization of UGT84A23 and UGT84A24. Overall, functional characterization and localization of UGT84A23 and UGT84A24 open up opportunities for further understanding the regulatory control of HT metabolism in plants and its coordination with other biochemical pathways in the metabolic network.

新規<i>C</i>-グルコシド合成法の開発とSGLT2阻害薬への応用—SGLT2阻害薬カナグリフロジンの創製—

新規<i>C</i>-グルコシド合成法の開発とSGLT2阻害薬への応用—SGLT2阻害薬カナグリフロジンの創製—

Dirigent Protein-Mediated Lignan and Cyanogenic Glucoside Formation in Flax Seed: Integrated Omics and MALDI Mass Spectrometry Imaging.

An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (-)- and (+)-pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis.

Cassava genome from a wild ancestor to cultivated varieties.

Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology.

Genomic organization of a UDP-glucosyltransferase gene determines differential accumulation of specific flavonoid glucosides in tepals

Glycosylation plays a major role in the chemical diversity of flavonoids. The wide diversity of the family-1 glycosyltransferase (UGT) impairs the determination of the biochemical function solely from its primary sequence. Here we combined differential expression and target metabolomic analysis in various Crocus species to identify a gene that is key in determining the flavonoid composition of Crocus species that belong to the Crocus series. UGT703B1 recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-3,7-O-diglucoside. These flavonols were present in C. sativus and C. cartwrightianus albus, both from series Crocus but absent in Crocus species from the other series analyzed. Further, the presence of both flavonols was associated with the expression of UGT703B1, and this expression was correlated with the presence of the UGT703B1 coding gene, with the exception of C. cancellatus, whose genomic sequence was present but contained a shorter intronic sequence and promoter alterations, suggesting the presence of regulatory sequences in the deleted part of that intron and promoter important for UGT703B1 expression. Overall, the data obtained supports the involvement of UGT703B1 in the formation of specific kaempferol and isorhamnetin glucosides, while demonstrating that the integration of metabolomic and differential expression analysis is a versatile tool for understanding a multigene family of UGTs in Crocus.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics

The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant).H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed.The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.