Profiling of secondary metabolites and DNA typing of three different Annona cultivars grown in Egypt.

OBJECTIVES: Natural products are often efficacious and safe alternatives to synthetic drugs. This study explored secondary leaves and bark metabolites profiles in extracts of a new Egyptian hybrid, Annona cherimola × Annona squamosa, known as Abdel Razek. This hybrid exhibited 100% similarity with A. cherimola as evidenced by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses. METHODS: Primary constituents in methanol extracts of different plant organs were identified. Extracts richest in alkaloids and polyphenolics were assessed for in vitro antioxidant activity and the most potent were further studied in vivo for treating gastric ulcer in rats. The latter activity was assessed histopathologically. RESULTS: Structural analysis with HPLC/ESI-MSn, and UPLC/HESI-MS/MS identified 63 metabolites, including seven amino acids, 20 alkaloids, 16 flavonoids, eight phenolics and other compounds. Severe stomach alteration was observed after ethanol induction in rats. Ulcer score, oxidative stress biomarkers, cell organelles biomarker enzymes, and gastrointestinal histological features improved to variable degrees after treatment with Annona Abdel Razek hybrid leaves and bark methanol extracts. CONCLUSION: Extracts of Annona Abdel Razek had showed in vitro antioxidant effect and may be promising for the treatment of gastric ulcers.

Phenolic Metabolites from Barley in Contribution to Phenome in soil Moisture Deficit.

Eight barley varieties from Europe and Asia were subjected to moisture deficit at various development stages. At the seedling stage and the flag leaf stage combined stress was applied. The experiment was designed for visualization of the correlation between the dynamics of changes in phenolic compound profiles and the external phenome. The most significant increase of compound content in water deficiency was observed for chrysoeriol and apigenin glycoconjugates acylated with methoxylated hydroxycinnamic acids that enhanced the UV-protection effectiveness. Moreover, other good antioxidants such as derivatives of luteolin and hordatines were also induced by moisture deficit. The structural diversity of metabolites of the contents changed in response to water deficiency in barley indicates their multipath activities under stress. Plants exposed to moisture deficit at the seedling stage mobilized twice as many metabolites as plants exposed to this stress at the flag leaf stage. Specific metabolites such as methoxyhydroxycinnamic acids participated in the long-term acclimation. In addition, differences in phenolome mobilization in response to moisture deficit applied at the vegetative and generative phases were correlated with the phenotypical consequences. Observations of plant yield and biomass gave us the possibility to discuss the developmentally related consequences of moisture deficit for plants' fitness.

Total Versus Inorganic and Organic Species of As, Cr, and Sb in Flavored and Functional Drinking Waters: Analysis and Risk Assessment.

Packing material can release certain elements such as As, Cr, or Sb into its content and, thus, contaminate the drinking water. The effect of As, Cr, and Sb on human health depends highly on the chemical species in which these elements are introduced into the body. For the above reasons quantification and speciation of As, Cr, and Sb in flavored and functional drinking water samples is an important issue. Total, inorganic, and organic species of As, Cr, and Sb including As(III), As(V), Cr(VI), Sb(III), and Sb(V) were studied in flavored and functional drinking waters. Analyses of total As, Cr, and Sb were conducted using inductively coupled plasma mass spectrometry (ICP-MS) according to ISO 17294-2:2016. The speciation analysis of arsenic, chromium, and antimony in bottled flavored and functional drinking waters was conducted with the use of the elemental (HPLC/ICP dynamic reaction cell (DRC) MS) and molecular (electrospray ionization MS/MS) mass spectrometry. Concentrations of total As, Cr, and Sb (µg∙L-1) in waters studied were in the ranges 0.0922 ± 0.0067 to 8.37 ± 0.52, 0.0474 ± 0.0014 to 1.310 ± 0.045, and 0.0797 ± 0.0026 to 1.145 ± 0.019, respectively. Speciation analysis showed that, apart from the toxic ionic species, known and unknown organic species were present in test samples. The risk assessment results proved that there is no risk associated with consumption of these tested beverages in terms of the non-carcinogenic effect of total and inorganic or organic species of As, Cr, and Sb.

The Effect of Different Water Extracts from Platycodon grandiflorum on Selected Factors Associated with Pathogenesis of Chronic Bronchitis in Rats.

The aim of this study was to assess the activity of extracts from Platycodon grandiflorum A. DC (PG) in a model of chronic bronchitis in rats. The research was carried out on three water extracts: E1 - from roots of field cultivated PG; E2 - from biotransformed roots of PG; E3 - from callus of PG. The extracts differed in saponins and inulin levels-the highest was measured in E3 and the lowest in E1. Identification of secondary metabolites was performed using two complementary LC-MS systems. Chronic bronchitis was induced by sodium metabisulfite (a source of SO2). Animals were treated with extracts for three weeks (100 mg/kg, intragastrically) and endothelial growth factor (VEGF), transforming growth factors (TGF-ß1, -ß2, -ß3), and mucin 5AC (MUC5AC) levels were determined in bronchoalveolar lavage fluid, whereas C reactive protein (CRP) level was measured in serum. Moreover, mRNA expression were assessed in bronchi and lungs. In SO2-exposed rats, an elevation of the CRP, TGF-ß1, TGF-ß2, VEGF, and mucin was found, but the extracts' administration mostly reversed this phenomenon, leading to control values. The results showed a strong anti-inflammatory effect of the extracts from PG.

Analytical Methods for Detection of Plant Metabolomes Changes in Response to Biotic and Abiotic Stresses.

Abiotic and biotic stresses are the main reasons of substantial crop yield losses worldwide. Research devoted to reveal mechanisms of plant reactions during their interactions with the environment are conducted on the level of genome, transcriptome, proteome, and metabolome. Data obtained during these studies would permit to define biochemical and physiological mechanisms of plant resistance or susceptibility to affecting factors/stresses. Metabolomics based on mass spectrometric techniques is an important part of research conducted in the direction of breeding new varieties of crop plants tolerant to the affecting stresses and possessing good agronomical features. Studies of this kind are carried out on model, crop and resurrection plants. Metabolites profiling yields large sets of data and due to this fact numerous advanced statistical and bioinformatic methods permitting to obtain qualitative and quantitative evaluation of the results have been developed. Moreover, advanced integration of metabolomics data with these obtained on other omics levels: genome, transcriptome and proteome should be carried out. Such a holistic approach would bring us closer to understanding biochemical and physiological processes of the cell and whole plant interacting with the environment and further apply these observations in successful breeding of stress tolerant or resistant crop plants.

Study on Speciation of As, Cr, and Sb in Bottled Flavored Drinking Water Samples Using Advanced Analytical Techniques IEC/SEC-HPLC/ICP-DRC-MS and ESI-MS/MS.

The main aim of the research was to develop a complementary analytical approach consisting of bespoke speciation analysis and non-targeted speciation analysis of As, Sb, and Cr in flavored bottled drinking water samples using HPLC/ICP-DRC-MS and ESI-MS/MS. The scope of two previously developed analytical procedures, (1) multielemental speciation procedure for AsIII, AsV, CrVI, SbIII, and SbV analysis and (2) arsenic speciation procedure for AsB, AsIII, DMA, MMA, and AsV quantification, was extended to the analysis of a new sample type in terms of bespoke speciation analysis. As for the non-targeted speciation, analysis size exclusion chromatography was used with ICP-MS and a complementary technique, ESI-MS/MS, was used for the organic species of As, Sb, and Cr screening. Full validation of procedures 1 and 2 was conducted. Procedure 1 and 2 were characterized with precision values in the range from 2.5% to 5.5% and from 3.6% to 7.2%, respectively. Obtained recoveries ranged from 97% to 106% and from 99% to 106% for procedures 1 and 2, respectively. Expanded uncertainties calculated for procedures 1 and 2 ranged from 6.1% to 9.4% and from 7.4% to 9.9%, respectively. The applicability of the proposed procedures was tested on bottled drinking water samples. Results for the real samples in procedure 1 were in the range from 0.286 ± 0.027 [µg L-1] to 0.414 ± 0.039 [µg L-1] for AsIII, from 0.900 ± 0.083 [µg L-1] to 3.26 ± 0.30 [µg L-1] for AsV, and from 0.201 ± 0.012 [µg L-1] to 0.524 ± 0.032 [µg L-1] for SbV. CrVI and SbIII were not detected in any sample. As for procedure 2, results were in the range from 0.0541 ± 0.0053 [µg L-1] to 0.554 ± 0.054 [µg L-1] for AsB. Results for AsIII and AsV obtained with procedure 2 were in good accordance with results obtained with procedure 1. DMA and MMA were not detected in any sample.

Drought-related secondary metabolites of barley (Hordeum vulgare L.) leaves and their metabolomic quantitative trait loci.

Determining the role of plant secondary metabolites in stress conditions is problematic due to the diversity of their structures and the complexity of their interdependence with different biological pathways. Correlation of metabolomic data with the genetic background provides essential information about the features of metabolites. LC-MS analysis of leaf metabolites from 100 barley recombinant inbred lines (RILs) revealed that 98 traits among 135 detected phenolic and terpenoid compounds significantly changed their level as a result of drought stress. Metabolites with similar patterns of change were grouped in modules, revealing differences among RILs and parental varieties at early and late stages of drought. The most significant changes in stress were observed for ferulic and sinapic acid derivatives as well as acylated glycosides of flavones. The tendency to accumulate methylated compounds was a major phenomenon in this set of samples. In addition, the polyamine derivatives hordatines as well as terpenoid blumenol C derivatives were observed to be drought related. The correlation of drought-related compounds with molecular marker polymorphisms resulted in the definition of metabolomic quantitative trait loci in the genomic regions of single-nucleotide polymorphism 3101-111 and simple sequence repeat Bmag0692 with multiple linkages to metabolites. The associations pointed to genes related to the defence response and response to cold, heat and oxidative stress, but not to genes related to biosynthesis of the compounds. We postulate that the significant metabolites have a role as antioxidants, regulators of gene expression and modulators of protein function in barley during drought.

Structural Characterization of Flavonoid Glycoconjugates and Their Derivatives with Mass Spectrometric Techniques.

Mass spectrometry is currently one of the most versatile and sensitive instrumental methods applied to structural characterization of plant secondary metabolite mixtures isolated from biological material including flavonoid glycoconjugates. Resolution of the applied mass spectrometers plays an important role in structural studies of mixtures of the target compounds isolated from biological material. High-resolution analyzers allow obtaining information about elemental composition of the analyzed compounds. Application of various mass spectrometric techniques, including different systems of ionization, analysis of both positive and negative ions of flavonoids, fragmentation of the protonated/deprotonated molecules and in some cases addition of metal ions to the studied compounds before ionization and fragmentation, may improve structural characterization of natural products. In our review we present different strategies allowing structural characterization of positional isomers and isobaric compounds existing in class of flavonoid glycoconjugates and their derivatives, which are synthetized in plants and are important components of the human food and drugs as well as animal feed.

Identification of Radiodegradation Products of Acebutolol and Alprenolol by HPLC/MS/MS.

Two therapeutically active compounds from the group of ß-blockers, acebutolol (AC) and alprenolol (AL), in solid form were subjected to ionizing radiation emitted by a beam of high energy electrons from an accelerator with a standard sterilization dose of 25 kGy and in higher doses of 50-400 kGy. The effects of irradiation were detected by chromatographic methods (TLC, HPLC) and a hyphenated method (HPLC/MS/MS). No significant changes in the physicochemical properties of both compounds studied irradiated with 25 kGy were noted, but upon irradiation with the highest dose (400 kGy) the loss of AC and AL content determined by HPLC was 2.79 and 9.12%, respectively. The product of AC decomposition and the two products of AL decomposition were separated and identified by HPLC/MS/MS. It has been established that radiodegradation of AC and AL takes place by oxidation, leading to formation of the products of radiolysis, most probably alcohol derivatives of the ß-blockers studied. The additional product that appears on radiodegradation of AL is probably formed as a result of two simultaneous reactions: oxidation and CH2 group elimination.

Comprehensive Tools of Alkaloid/Volatile Compounds-Metabolomics and DNA Profiles: Bioassay-Role-Guided Differentiation Process of Six Annona sp. Grown in Egypt as Anticancer Therapy.

Trees of the Annona species that grow in the tropics and subtropics contain compounds that are highly valuable for pharmacological research and medication development and have anticancer, antioxidant, and migratory properties. Metabolomics was used to functionally characterize natural products and to distinguish differences between varieties. Natural products are therefore bioactive-marked and highly respected in the field of drug innovation. Our study aimed to evaluate the interrelationships among six Annona species. By utilizing six Start Codon Targeted (SCoT) and six Inter Simple Sequence Repeat (ISSR) primers for DNA fingerprinting, we discovered polymorphism percentages of 45.16 and 35.29%, respectively. The comparison of the profiles of 78 distinct volatile oil compounds in six Annona species was accomplished through the utilization of GC-MS-based plant metabolomics. Additionally, the differentiation process of 74 characterized alkaloid compound metabolomics was conducted through a structural analysis using HPLC-ESI-MSn and UPLC-HESI-MS/MS, and antiproliferative activities were assessed on five in vitro cell lines. High-throughput, low-sensitivity LC/MS-based metabolomics has facilitated comprehensive examinations of alterations in secondary metabolites through the utilization of bioassay-guided differentiation processes. This has been accomplished by employing twenty-four extracts derived from six distinct Annona species, which were subjected to in vitro evaluation. The primary objective of this evaluation was to investigate the IC50 profile as well as the antioxidant and migration activities. It should be noted, however, that these investigations were exclusively conducted utilizing the most potent extracts. These extracts were thoroughly examined on both the HepG2 and Caco cell lines to elucidate their potential anticancer effects. In vitro tests on cell cultures showed a significant concentration cytotoxic effect on all cell lines (HepG2, HCT, Caco, Mcf-7, and T47D) treated with six essential oil samples at the exposure time (48 h). Therefore, they showed remarkable antioxidant activity with simultaneous cytotoxic effects. In total, 50% and 80% of the A. muricata extract, the extract with the highest migratory activity, demonstrated a dose-dependent inhibition of migration. It was strong on highly metastatic Caco cells 48 h after treatment and scraping the Caco cell sheet, with the best reduction in the migration of HepG2 cells caused by the 50% A. reticulata extract. Also, the samples showing a significant IC50 value showed a significant effect in stopping metastasis and invasion of various cancer cell lines, making them an interesting topic for further research.

Separation of Chromatographic Co-Eluted Compounds by Clustering and by Functional Data Analysis.

Peak overlapping is a common problem in chromatography, mainly in the case of complex biological mixtures, i.e., metabolites. Due to the existence of the phenomenon of co-elution of different compounds with similar chromatographic properties, peak separation becomes challenging. In this paper, two computational methods of separating peaks, applied, for the first time, to large chromatographic datasets, are described, compared, and experimentally validated. The methods lead from raw observations to data that can form inputs for statistical analysis. First, in both methods, data are normalized by the mass of sample, the baseline is removed, retention time alignment is conducted, and detection of peaks is performed. Then, in the first method, clustering is used to separate overlapping peaks, whereas in the second method, functional principal component analysis (FPCA) is applied for the same purpose. Simulated data and experimental results are used as examples to present both methods and to compare them. Real data were obtained in a study of metabolomic changes in barley (Hordeum vulgare) leaves under drought stress. The results suggest that both methods are suitable for separation of overlapping peaks, but the additional advantage of the FPCA is the possibility to assess the variability of individual compounds present within the same peaks of different chromatograms.

Comprehensive metabolomic, lipidomic and pathological profiles of baobab (Adansonia digitata) fruit pulp extracts in diabetic rats.

Baobab fruit pulp Adansonia digitata (AD) has received attention due to its numerous nutritional and medicinal values. In the current study, tentative identification was performed due to limited information available on its phytochemical composition. Phytochemicals from AD fruit pulp were obtained using successive organic solvent fractionation. The LC-MSMS analysis led to identification of 91 metabolites from methanol, butanol and ethyl acetate extracts. Moreover, 20 compounds were identified in the petroleum ether extract based on high resolution ion masses. In vitro antidiabetic and antioxidant properties of selected extracts were investigated using enzyme activity and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, respectively. Biological screening of the antidiabetic effects of target extracts was performed against streptozotocin-induced diabetes in experimental animals, following daily oral treatment for 3 successive weeks. Serum glucose, insulin, adiponectin, superoxide dismutase (SOD), lipid peroxide, cholesterol and HDL levels were measured. Finally, histopathological and immunohistochemical examinations of pancreas were carried out. Results revealed that animal groups treated daily with butanol (BuOH) and petroleum ether extracts of AD (oil) exhibited a significant improvement in carbohydrate and lipid metabolism as well as antioxidant effect. Both extracts revealed superior effects with respect to the total (TT) and ethyl acetate (EtOAc) extracts. Histopathological and immunohistochemical findings supported these results, showing marked protection of the pancreas. Thus, baobab oil and butanolic extract of the fruit pulp protected animals against STZ-induced diabetic changes, in addition to attenuation of lipid peroxidation, hypercholesterolemia and oxidation.

LC-MSMS profiling of flavonoid conjugates in wild Mexican lupine, Lupinus reflexus.

Profiles of flavonoid conjugates present in the root and leaf tissues of the Mexican wild lupine, Lupinus reflexus, were established using two LC-MSMS systems in the positive and negative ion modes. The ion trap mass spectrometer and quadrupole time-of flight instrument provided sequential MS(n) spectra and MSMS spectra with accurate m/z values of [M + H](+) and [M - H] (-) ions, respectively. Sixty-two flavone and isoflavone glycoconjugates were found and tentatively identified. Numerous isomeric or isobaric compounds with the same molecular mass could be differentiated. Isomeric di- and mono glucosides of biochanin A, genistein, 2'-hydroxygenistein, luteone, and 2,3-didehydrokievitone were distinguished on the basis of relative abundances of product ions. The studied flavonoid glycoconjugates were acylated with dicarboxylic aliphatic acids and their methyl esters at either the aglycone or glycosidic moiety.

Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula.

INTRODUCTION: Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono- or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. METHODOLOGY: Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry. RESULTS: The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p-coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high-resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. PERSPECTIVES: The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species.

Arsenic species and their transformation pathways in marine plants. Usefulness of advanced hyphenated techniques HPLC/ICP-MS and UPLC/ESI-MS/MS in arsenic species analysis.

The growing popularity of algae as a foodstuff around the world raises concern for the safety of this food type with respect to arsenic content in algae. The need for determination of total arsenic content and arsenic speciation in algae food has become an important issue. In this paper we have developed a complete analytical procedure for arsenic determination in algae products comprised of 1) total arsenic (tAs) determination in native algae samples after digestion, 2) extraction of As species with the use of two extraction methods with three extracting agents, 3) extracted total arsenic (extracted tAs) determination in algae extracts, 4) bespoke As speciation, 4) mass balance estimation based on extracted tAs and bespoke As speciation results, 5) unknown arsenic (uAs) species identification. Two advanced hyphenated techniques, HPLC/ICP-MS and UPLC/ESI-MS/MS, were employed along with the HPLC/ICP-MS method validation. Total As content in edible algae samples was found to range from (19.28 ± 0.45) mg kg-1 up to (72.6 ± 2.7) mg kg-1. Bespoke arsenic speciation of edible algae samples has revealed the presence of some known inorganic and simple organic As compounds such as As(III) from

Silver nanoparticles affect phenolic and phytoalexin composition of Arabidopsis thaliana.

Silver nanoparticles are widely used in industry, medicine, biotechnology and agriculture. As a consequence, these nanoparticles are reaching the environment as waste products, which might have a negative impact on the environment, especially on plants. This includes the elicitation of various biochemical processes in plants. In this article, we report on the changes in secondary metabolic profile of Arabidopsis thaliana seedlings subjected to silver nanoparticle treatment in vitro. Briefly, various sizes (10 nm, 40 nm and 100 nm in diameter) and concentrations (0.5-5.0 ppm) of silver nanoparticles were tested. Ultraperformance liquid chromatography coupled with ultraviolet and fluorescence detectors as well as hyphenated to a high-resolution mass spectrometer (UPLC-PDA-FLR, UPLC-HESI-HRMS) and HPLC - ion trap mass spectrometer (HPLC-ESI-MS/MS), were applied to identify and quantify secondary metabolites. To understand whether silver ions could induce changes in the secondary metabolite profile, seedlings treated with silver nitrate in concentrations equivalent to these of nanoparticles were also analysed. The results showed significant differences in the accumulation of phenolic and indole compounds between treatments. Silver nanoparticles and silver ions induced the biosynthesis of camalexin, hydroxycamalexin O-hexoside and hydroxycamalexin malonyl-hexoside. These compounds are important phytoalexins for Brassicaceae family (especially for Camelinae clad) and are also synthetized in response to biotic and abiotic stresses. Statistically significant changes have been also observed for five phenolic compounds and 5'Glucosyl-dihydroneoascorbigen in different treatment conditions.

Chemical characterization and in vivo antioxidant activity of parsley (Petroselinum crispum) aqueous extract.

Twenty-nine flavonoid glycosides were identified in the aqueous extract (PC) of Petroselinum crispum var. crispum leaves and apiin, the major compound, was isolated thereof. Apigenin was obtained (90% purity) by the hydrolysis of apiin. A high content of phenolics (12.49 ± 1.70 mg GAE per g of parsley extract - Folin-Ciocalteu method) and total flavonoids (15.05 ± 2.20 mg of quercetin equivalents per g of parsley extract - aluminum chloride method) was quantified in P. crispum, as well as high antioxidant activity ((EC50 - 15.50 mg mL-1, DPPH method) and (189.8 mM Fe(ii) per mg of dry plant aqueous extract - FRAP method)). In vivo analysis with Saccharomyces cerevisiae cells showed low toxicity of the aqueous extract of parsley, however, it revealed a high dose-dependent antioxidant potential, mainly in the lipoperoxidation assay. In addition, flavonoid apiin also showed antioxidant action on yeast cells under oxidative stress in the cell viability assay (0.1 mM) and lipid peroxidation (0.01 and 0.1 mM), while apigenin was slightly antioxidant. Therefore, it is likely that the antioxidant activity of apiin is related to the total antioxidant capacity of parsley.

Retraction: Organometallic Ag nanostructures prepared using Hypericum perforatum extract are highly effective against multidrug-resistant bacteria.

[This retracts the article DOI: 10.1039/C8RA05655B.].

Correction: Marslin, G., et al. Secondary Metabolites in the Green Synthesis of Metallic Nanoparticles. Materials 2018, 11, 940.

The authors have overlooked a few mistakes when rearranging the Table 1 and Table 2 and references at the final stages, which were carried-over to the published version of the review [...].

Bioaccessibility of defatted lupin seed phenolic compounds in a standardized static in vitro digestion system.

Phenolic compounds are secondary plant metabolites whose beneficial health effects make them of intense interest to researchers. The aim of the study presented here was to evaluate the change in the phenolic compound profile of lupin seed in in vitro digestion. The most abundant phenolic compounds in undigested lupin seeds were mostly apigenin derivatives. The in vitro digestion of lupin seeds resulted in qualitatively altered phenolic compound profiles. Approximately 80% of phenolic compounds were released from lupin seeds during the in vitro digestion, which simulated gastric processes. Continued digestion, imitating the intestinal phase, additionally increased the bioaccessibility of lupin seed polyphenols by about 10%. The in vitro gastrointestinal model was also used to elucidate how the content of native phenolic compounds affects the digestion susceptibility of lupin seed proteins. An inverse correlation between protein digestibility and phenolic compound content, was also demonstrated.