Low Molecular Weight Polysaccharides Research Articles

Cell Wall Polysaccharides of Near-Isogenic Lines of Melon (Cucumis melo L.) and Their Inbred Parentals Which Show Differential Flesh Firmness or Physiological Behavior

We characterized differences in cell wall material and polysaccharide structures, due to the quantitative trait loci associated with higher flesh firmness in a nonclimacteric near-isogenic line (NIL) SC7-2, and with the climacteric behavior of the NIL SC3-5-1, using their nonclimacteric inbred parentals, "Piel de Sapo" (PS) and PI 161375 (SC). PS was firmer and had a higher ripening index and greater hemicellulosic content than SC, with its lower wall material yield, and uronic acid, neutral sugar, cellulose and free sugar content and higher pectic content. SC3-5-1 showed lower uronic acid values, a higher soluble solid content, and similar flesh firmness to PS. SC3-5-1 yielded mainly high molecular weight polysaccharides in the imidazole-soluble fraction than PS. SC7-2 showed greater flesh firmness, a higher neutral sugar (especially galactose and mannose) and uronic acid content, together with a larger cellulose and α-cellulose residue than PS. SC7-2 also contained more polysaccharides of low molecular weight in the first pectic fraction and shifted toward higher molecular weights in the main peak of the 4 M potassium-soluble fraction compared with PS.

Promotion of fungal growth and underlying physiochemical changes of polysaccharides in Rigidoporus ulmarius, an edible Basidiomycete mushroom

Rigidoporus ulmarius is an edible fungus used for medicinal purposes. The aim of this research was to investigate the effects of different carbon sources and initial pH values of the medium on mycelial growth, and analyze underlying changes in polysaccharides. The maximum mycelial growth of 20.91g/l was obtained at 20g/l potato dextrose broth (PDB) with 20g/l glucose, pH 4.5. Increasing sucrose- or glucose concentration from 5 to 20g/l in the media enhanced mycelia synthesizing polysaccharides in the molecular-weight range (>600kDa), and inhibited that of polysaccharide in molecular-weight range (5–20kDa). When the initial pH of the medium increased from 4.5 to 6.5, mycelia synthesized polysaccharides in high-molecular-weight (>600kDa) decreased and that of low-molecular weight (8–11kDa) polysaccharides increased. Feeding with sucrose or glucose resulted in a direct dosage effect to the fucose, glucose, and mannose components of the polysaccharides.

Hydrogen rich gas production by thermocatalytic decomposition of kenaf biomass

Kenaf ( Hibiscus cannabinus L.), a well known energy crop and an annual herbaceous plant grows very fast with low lodging susceptibility was used as representative lignocellulosic biomass in the present work. Thermocatalytic conversions were performed by aqueous phase reforming (APR) of kenaf hydrolysates and direct gasification of solid biomass of kenaf using 5% Pt on activated carbon as catalyst. Hydrolysates used in APR experiments were prepared by solubilization of kenaf biomass in subcritical water under CO 2 gas pressure. APR of kenaf hydrolysate with low molecular weight polysaccharides in the presence of the reforming catalyst produced more gas compared to the hydrolysate that had high molecular weight polysaccharides. APR experiments of kenaf biomass hydrolysates and glucose, which was used as a simplest biomass model compound, in the presence of catalyst produced various amounts of gas mixtures that consisted of H 2, CO, CO 2, CH 4 and C 2H 6. The ratios of H 2 to other gases produced were 0.98, 1.50 and 1.35 for 150 °C and 250 °C subcritical water-treated kenaf hydrolysates and glucose, respectively. These ratios indicated that more the degraded organic content of kenaf hydrolysate the better selectivity for hydrogen production. Although APR of 250 °C-kenaf hydrolysate resulted in similar gas content and composition as glucose, the gas volume produced was three times higher in glucose feed. The use of solid kenaf biomass as starting feedstock in APR experiments resulted in less gas production since the activity of catalyst was lowered by solid biomass particles.

Structural characterization and hydroxyl radicals scavenging capacity of a polysaccharide from the fruiting bodies of Auricularia polytricha

Abstract One low molecular-weight polysaccharide (APP) of 2.8 × 10 4 Da was isolated from the fruiting body of Auricularia polytricha . Partial hydrolysis with acid, periodate oxidation–Smith degradation, acetylation, methylation analysis and NMR spectroscopy ( 1 H, 13 C) were conducted to elucidate its structure and the repeating unit of APP was established as: the backbone was composed of (1 → 3)-linked-β- d -mannopyranosyl, (1 → 3)-linked-β- d -galactopyranosyl and (1 → 3,6)-linked-β- d -mannopyranosyl residues in the ratio of 3:2:1, and terminated with one single terminal (1→)-β- d -glucopyranosyl at the O -6 position of (1 → 3,6)-linked-β- d -mannopyranosyl along the main chain. Preliminary antioxidant activity test in vitro showed APP could potentialize scavenging effect on hydroxyl radicals in a dose dependent manner.

Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by beta-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 degrees C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0-5.5. They were stable in the pH range 5.0-10.0 and 5.5-8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55 and 60 degrees C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 degrees C.

Genetic Analysis of the rkp-3 Gene Region in Sinorhizobium meliloti 41: rkpY Directs Capsular Polysaccharide Synthesis to KR5 Antigen Production

Rhizobial surface polysaccharides, including capsular polysaccharides (KPS), are involved in symbiotic infection. The rkp-3 locus of Sinorhizobium meliloti 41 is responsible for the production of pseudaminic acid, one of the components of the KR5 antigen, a strain-specific KPS. We have extended the sequence determination and genetic dissection of the rkp-3 region to clarify the structure and function of the rkpY gene and to identify additional rkp genes. Except for rkpY, no other genes were found where mutation affected the KPS structure and symbiosis. These mutants show a unique phenotype producing a low molecular weight polysaccharide (LMW PS). Creating double mutants, we have shown that biosynthesis genes of the KR5 antigen except rkpZ are not necessary for the production of this LMW PS. Polysaccharide analysis of genetically modified strains suggests that rkpY has pleiotropic effects on polysaccharide production. It directs KPS synthesis to the KR5 antigen and influences lipo-oligo 3-deoxy-d-manno-2 octulosonic acid (Kdo) production in S. meliloti 41. In addition, rkpY suppresses the lipo-oligoKdo production when it is introduced into S. meliloti 1021.

A polysaccharide isolated from Agaricus blazei Murill inhibits sialyl Lewis X/E-selectin-mediated metastatic potential in HT-29 cells through down-regulating α-1,3-fucosyltransferase-VII (FucT-VII)

In this research, one low molecular weight polysaccharide (LMW-ABP) was isolated from the fruiting bodies of Agaricus blazei Murill. We reported that the effect of LMW-ABP on inhibiting the interaction between E-selectin and sialyl Lewis X (sLe x) by flow cytometry and real-time reverse transcription polymerase chain reaction (RT-PCR) technology. Different concentrations of LMW-ABP (5, 10, and 20 mg/L) could effectively dose-dependently inhibit adhesion of HT-29 cells to human umbilical vein endothelial cells (HUVECs) in static conditions, as well as down-regulating the expression of both α-1,3-fucosyltransferase-VII (FucT-VII) and sLe x in transcription or translation level, respectively. These results suggest that LMW-ABP could suppress the metastasizing capacity of cancer cells through interfering with the interaction between E-selectin and sLe x. Thus based on these findings, LMW-ABP is expected to be having enormous potential for use in treatment for neoplasm metastasis.

Fate and impact of organics in an immersed membrane bioreactor applied to brine denitrification and ion exchange regeneration

The application of membrane bioreactors (MBRs) to brine denitrification for ion exchange regeneration has been studied. The developed culture was capable of complete brine denitrification at 50 gNaCl.l−1. Denitrification reduced to c.60% and c.70% when salinity was respectively increased to 75 and 100g.l−1, presumed to be due to reduced growth rate and the low imposed solids retention time (10 days). Polysaccharide secretion was not induced by stressed cells following salt shocking, implying that cell lysis did not occur. Fouling propensity, monitored by critical flux, was steady at 12–15l.m−2.h−1 during salinity shocking and after brine recirculation, indicating that the system was stable following perturbation. Low molecular weight polysaccharide physically adsorbed onto the nitrate selective anion exchange resin during regeneration reducing exchange capacity by c.6.5% when operating up to complete exhaustion. However, based on a breakthrough threshold of 10 mgNO3−-N.l−1 the exchange capacity was comparative to that determined when using freshly produced brine for regeneration. It was concluded that a denitrification MBR was an appropriate technology for IEX spent brine recovery and reuse.

A novel water-soluble β-(1→6)- d-glucan isolated from the fruit bodies of Bulgaria inquinans (Fries)

A low molecular-weight polysaccharide named BIWP2 was purified from the fruit bodies of Bulgaria Inquinans (Fries) via hot-water extraction, followed by freeze-thawing and gel filtration chromatography on Sephadex G-75. Monosaccharide composition analysis revealed that BIWP2 contained exclusively glucose. High performance size exclusion chromatography (HPSEC) showed that it was a homogeneous polysaccharide fraction. Its molecular weight was estimated to be 2.6 KD and the polydispersity index ( M w/ M n) was calculated to be 1.4. Periodate oxidation, methylation, and NMR analyses indicated that BIWP2 was a linear β-(1→6)- d-glucan without side chains. This is the first time to report a linear β-(1→6)- d-glucan with low molecular weight in non-lichenized ascomycete.

In Vitro Antioxidant Activities of Low-Molecular-Weight Polysaccharides with Various Functional Groups

The objectives of this study were to evaluate the effect of different functional groups of sulfate, amine, and hydroxyl and/or their ionized groups on in vitro antioxidant capacities of low-molecular-weight polysaccharides (LMPS) prepared from agar (LMAG), chitosan (LMCH), and starch (LMST), respectively, and to elucidate their structure-activity relationship. Ascorbic acid and ethylenediaminetetraacetic acid (EDTA) were used as positive controls. The in vitro antioxidant capacities of LMAG and LMCH were higher than that of LMST in the DPPH radical, superoxide radical, hydrogen peroxide, and nitric oxide radical scavenging and ferrous metal-chelating capacities. The different scavenging capacities may be due to the combined effects of the different sizes of the electron-cloud density and the different accessibility between free radical and LMPS, which, in turn, depends upon the different hydrophobicities of the constituent sugars.

A001 Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets

P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized Pselectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC50 of 20 nM as compared to 400 nM for heparin and >25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized Pselectin with a KD of 1.2 nM, two orders of magnitude greater than the KD of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

A Low Molecular Weight Polysaccharide Isolated from Agaricus blazei Murill (LMPAB) Exhibits Its Anti-Metastatic Effect by Down-Regulating Metalloproteinase-9 and Up-Regulating Nm23-H1

The components of Agaricus blazei Murill (AbM) have been shown to possess antitumor potentials. Herein, we attempted to explore the anti-metastatic effect and underlying mechanism of a low molecular weight polysaccharide isolated from AbM (LMPAB). Matrigel invasion assay was applied to evaluate the effect of LMPAB on migration of BEL-7402 hepatic cancer cells in vitro. In vivo, the anti-metastatic effect of LMPAB was investigated in mouse B16 melanoma and a double-grafted SW180 tumor models. mRNA and protein levels of metalloproteinase-9 (MMP-9) or nm23-H1 upon LMPAB treatment were detected by real-time PCR and immunohistochemistry assays. LMPAB significantly reduced the invasion of BEL-7402 cells. In vivo, LMPAB was revealed to decrease lung metastatic foci in mouse B16 melanoma model. In the double-grafted SW180 mouse tumor model, we further demonstrated that intratumoral treatment of LMPAB inhibited the growth of tumor on treated side but also suppresses the regression of metastatic tumors on the non-treated side. Moreover, LMPAB reduced MMP-9 but enhanced nm23-H1 mRNA and protein expression. LMPAB displays anti-metastatic activities, indicating the potential of its clinical application for the prevention and treatment of cancer metastasis. Its anti-metastatic effect may relate to the modulation on MMP-9 and nm23-H1.

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets

Background: P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Methods: Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. Results: The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC 50 of 20 nM as compared to 400 nM for heparin and < 25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the K D of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. General significance: Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.

The use of very low molecular weight polysaccharides as depressants in PGM flotation

Polysaccharides traditionally used in PGM flotation have molecular weights (MW) varying from 150,000 to 600,000 as measured using gel permeation columns (GPC). In recent years polysaccharides at the lower end of the range (MW of 100,000–150,000) have been used effectively. This work evaluates the use of a modified starch depressant with a much lower average MW of 20,000 as a depressant and compares its performance on two Merensky ores to that of depressants currently used in industry, namely guar gum and carboxymethylcellulose (CMC). The UCT batch flotation system was used to assess the performance of the starch as a depressant of naturally floatable gangue (NFG) as well as its effect on sulphide mineral recovery at low and high dosages. Determination of the amount of polymer adsorbed was also done by assessing polymer in solution at various concentrations using the Du Bois method. PGM assays were carried out on one ore sample. Results indicated that this starch is an effective depressant of NFG.

Structural analysis of the polysaccharides from Echinacea angustifolia radix

In this paper we report the characterization by monosaccharide and linkage analyses and by NMR spectroscopy and size exclusion chromatography of the carbohydrate fraction extracted from Echinacea angustifolia radix. In addition, the products of endo-pectin lyase, endo-pectate lyase, endo-polygalacturonase, endo-galactanase, and endo-arabinase digestion were characterized by MALDI-TOF mass spectrometry. The data obtained during this study showed that the carbohydrate fraction extracted from E. angustifolia radix is constituted by two polysaccharides with molecular weight of about 128,000 and 4500 Da. The low molecular weight polysaccharide corresponds to inulin while the high molecular weight component is a high metoxy pectin in which the backbone structure of the smooth region is constituted by α-(1–4)-polygalacturonan partially methyl esterified (60%) and acetylated (9%) and with the hairy regions containing 2- O- and 2,4- O-rhamnopyranose, 5- O- and 3,5- O-arabinofuranose, 3,6-galactopyranose, and terminal rhamnopyranose, arabinofuranose, arabinopyranose, galactopyranose, and galacturonopyranose. Mass spectrometry data of a galactanase treated sample showed evidence of a novel structure in the pectin hairy region, namely a galactose–galacturonic acid alternating sequence.

Antioxidant effect of Inonotus obliquus

The mushroom Inonotus obliquus (Fr.) Pilát (Hymenochaetaceae), has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The purpose of this study was to elucidate the antioxidant capacities of Inonotus obliquus. Four extracts from the fungus were evaluated for antioxidant activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, and peroxyl radicals. The polyphenolic extract had a strong antioxidant activity, and the extract containing triterpenoids and steroids presented a relatively strong antioxidant effect. The polysaccharide extract, however, was inactive. The protective effects of these four extracts were assessed against hydrogen peroxide-induced oxidative stress using a human keratinocyte cell line, HaCaT. Our results show that the polyphenolic extract protected these cells against hydrogen peroxide-induced oxidative stress, while the polysaccharide, triterpenoid and steroid extracts were ineffective. Additionally, the remnant polyphenolic and low molecular weight polysaccharide extracts showed a weakly protective effect at a concentration of 50 μg/ml. Our results indicate that Inonotus obliquus has the capacity to scavenge free radicals at concentrations higher than 5 μg/ml and that the polyphenolic extract can protect cells against oxidative stress.

Pulse NMR Study of Refrigerated Cooked Rice and Comparison with Physicochemical Properties

The behavior of water molecules in refrigerated cooked rice was examined by measuring spin-spin relaxation time T2 using a pulse NMR spectrometer. T2 decay curve was analyzed using two-component and non-linear regression analysis and the presence of three kinds of water components with different T2 were revealed. In the case of fresh cooked rice, the observed T2 and the relative population were 0.9 ms (short T2) and 13%, 26 ms (intermediate T2) and 76%, and 200 ms (long T2) and 11%, respectively. The short T2 component was ascribed to the water molecules hydrated directly on the starch molecules. Intermediate T2 component was dominant and ascribed to the water molecules accommodated in the starch matrix inside the rice grain. The mobility of water molecules was suppressed remarkably during the storage at 0°C while it was almost independent of the refrigerated storage below −18°C. The long T2 component was ascribed to those grains accommodated in the surface luster film that was formed in the initial stage of the cooking process and composed of low molecular weight polysaccharides. The relaxation property observed by NMR was well consistent with the physicochemical properties.

BIOCHEMICAL STUDIES RELATED TO TEXTURAL REGULATION DURING RIPENING OF BANANA AND MANGO FRUIT

The changes occurring during ripening of Cavendish banana and Alphonso mango were studied, with emphasis on carbohydrates and the carbohydrate hydrolases responsible for textural softening. In both banana and mango there was a significant increase in soluble sugars, mainly in the reducing types. There was a gross increase in uronides and a steady decrease in starch, cellulose, hemicellose and pectins during each stage of ripening. The new observation made in the present study is that xylanase and laminarinase among the glycanases, and α-mannosidase followed by β-galactosidase among glycosidases, appeared to play a crucial role in textural softening of banana. Similarly, in mango, glycanases such as mannanase, galactanase and arabananase were recognised as the key enzymes for textural degradation. There was a major drop in molecular weights of carbohydrate polymers during ripening of mango with the complete disappearance of low molecular weight polysaccharides of below 70,000 daltons. This information is useful for monitoring, at the biochemical level, the textural loss during ripening of banana and mango without inhibiting ethylene-associated natural ripening.

Purification and characterization of antithrombotics from Syzygium aromaticum (L.) MErr. & PERRY.

Two antithrombotic polysaccharides with relatively high molecular weight (HMW) and low molecular weight (LMW) were isolated from the flower buds of Syzygium aromaticum (L.) MERR. & PERRY (clove) by anion-exchange chromatography, hydrophobic interaction column chromatography and size exclusion chromatography (LMW: EC-2B-IIIa-2, M.W. ca. 34000; HMW: EC-2C-Ia-2, M.W. ca. 103000). The LMW polysaccharide was mainly composed of Rha, Gal, GalA and Ara (molar %: 24.1, 18.9, 18.0 and 17.9, respectively) with 10.8% of sulfate and 18.2% of protein. The HMW fraction consisted of Ara, Gal, Glc and Rha (molar %: 26.0, 23.7, 17.5 and 12.4, respectively) with 15.4% of sulfate and 8.0% of protein. Both polysaccharides had the backbone of type I rhamnogalacturonan and the side chain of arabinan. Also, most of the sulfates were attached at the position 6 of 3-linked galactosyl residues. Compared to the antithrombotic activity of the HMW fraction (plasma clotting time of 145 s in APTT assay), the LMW fraction displayed a slightly low activity (90 s). However, animal studies indicated that crude LMW polysaccharide did not show acute toxicity, while the acute LD50 of the HMW fraction was approximately 2-fold lower than that of heparin.

Iron Supplementation in Preterm Infants: A Study Comparing the Effect and Tolerance of a Fe2+ and a Nonionic FeIII Compound

The more widely used divalent forms of iron (Fe2+) supplementation often lead to gastrointestinal symptoms in preterm infants although little is known about the use of nonionic trivalent iron preparations (FeIII) in these patients. It is especially under this nonionic form that dietary iron is available. For this reason, a randomized controlled study was undertaken to compare the efficacy and the extent of possible side effects in two groups of preterm infants. In one group, the elemental iron was given in the Fe2+ form, while the other group received a nonionic trivalent iron (FeIII) complexed with polysaccharides of low molecular weight. Both groups received 7.5 mg elemental iron daily. Measured parameters in the two study groups did not differ significantly throughout the study period of 14 weeks. Both forms of iron supplementation were well tolerated. However, vomiting, diarrhea, or constipation occurred slightly more often in the group receiving iron supplementation in the Fe2+ form without reaching statistical difference. The authors found a nonionic trivalent polysaccharide-iron complex given as iron supplementation as effective as the generally more favored ferrous sulphate. Since iron therapy is mandatory in the preterm infant, the use of trivalent iron complexes can be considered as a good alternative.