Pectolinarigenin, an aglycone of pectolinarin, has more potent inhibitory activities on melanogenesis than pectolinarin.

Pectolinarin and pectolinarigenin have been reported to be major compounds in Cirsium setidens. In the present study, we demonstrated inhibitory effects of pectolinarin and pectolinarigenin from C. setidens on melanogenesis. Melanin synthesis was decreased in both pectolinarin- and pectolinarigenin-treated melan-a cells and in a reconstructed human skin model. However, pectolinarigenin treatment showed more potent inhibitory activity of melanin synthesis than did pectolinarin treatment. The concentrations of pectolinarin and pectolinarigenin in C. setidens water extracts were determined by HPLC. Unfortunately, the amount of pectolinarigenin of C. setidens water extract was lower than that of pectolinarin. To increase the pectolinarigenin content in C. setidens water extract, several component conversion methods were studied. Consequently, we identified that microwave irradiation under 1% acetic acid was an optimum sugar elimination method.

Composition and Bioactivity of Lipophilic Metabolites from Needles and Twigs of Korean and Siberian Pines (Pinus koraiensis Siebold & Zucc. and Pinus sibirica Du Tour).

Lipophilic extractive metabolites in different parts of the shoot system (needles and defoliated twigs) of Korean pine, Pinus koraiensis, and Siberian pine, Pinus sibirica, were studied by GC/MS. Korean pine needles comprised mainly bornyl p-coumarate, heterocyclic 15-O-functionalized labdane type acids (lambertianic acid), 10-nonacosanol, sterols and their esters. While Siberian pine needles contained less bornyl p-coumarate, lambertianic acid, sterols and their esters, but were richer in other 15-O-functionalized labdane type acids. The major components of the twig extract of P. koraiensis were lambertianic acid, abietane and isopimarane type acids, cembrane type alcohols, 8-O-functionalized labdanoids, sterols, sterol esters, and acylglycerols. The same extract of P. sibirica differed in larger amounts of other 15-O-functionalized labdane type acids and pinolenic acid glycerides, but in less quantities of cembranoids and 8-O-functionalized labdanoids. The labdane type pinusolic acid was detected for the first time in Korean pine. P. koraiensis was found to be unique in the genus for an ability to synthesize phyllocladane diterpenoids. The content of bound Δ5 -unsaturated polymethylene-interrupted fatty acids in the twig extracts of the both pines was similar or superior to that in their seed oil. Among the pines' metabolites tested isocembrol was strongest in inhibition of both α-glucosidase (IC50 2.9 µg/ml) and NO production in activated macrophages (IC50 3.6 µg/ml).

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

Chalcones from the flowers of Coreopsis lanceolata and their in vitro antioxidative activity.

Three new chalcones, 3,2'-dihydroxy-4,3'-dimethoxychalcone-4'-glucoside (1), 4'-O-(2'''-O-caffeoyl)2',3',3,4-tetrahydroxychalcone (2), and 2',4',3-trihydroxy-3',4-dimethoxychalcone (3), along with five known phenolics, were isolated from Coreopsis lanceolata flowers. The structures of the new compounds were elucidated by extensive spectroscopic methods including NMR and MS. The three new chalcones showed a good in vitro HepG2 cell protecting effect against tert-butylhydroperoxide-induced oxidative stress.

Lipophilic extracts from needles and defoliated twigs of Pinus pumila from two different populations.

Hexane extracts of needles and defoliated twigs of Pinus pumila (Pall.) Regel from two distant populations, located in the southwest and the east (i.e., Lake Baikal region and Sakhalin Island) of the species distribution range were studied by GC/MS analysis. Composition and retention indices of major components were determined. A drastic composition divergence for the extracts of P. pumila needles and defoliated twigs, depending on growth location, was established. Needle extracts from the eastern population sample contained mainly labdane-type acids (anticopalic acid derivatives), whereas the predominant components of needle extracts from the other population sample were abietane-type acids (abietic, neoabietic acids) and isopimarane-type diterpenoids (sandaracopimaric acid, sandaracopimaradien-3ß-ol). The main components of defoliated twig extracts from Sakhalin Island population sample were abietane-type acids and cembrane-type diterpenoids, while content of these compounds in the extracts of the southwestern marginal population sample was remarkably lower.

Rapid identification and evaluation of antioxidant compounds from extracts of Petasites japonicus by hyphenated-HPLC techniques.

Gradient HPLC coupled to Diode Array Detector (DAD), MS/MS and NMR was applied to the rapid structure determination of major compounds of methanol extracts from leaves and roots of Petasites japonicus. The relative antioxidant capacities of the compounds were evaluated by an HPLC system with post-column on-line antioxidant detection based on 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging. Six compounds were successfully separated on a reverse-phase C(18) column and were identified as 5-caffeoylquinic acid (5-CQA), fukinolic acid (FA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), quercetin-3-O-(6″-acetyl)-ß-glucopyranoside (QAG), 4,5-di-O-caffeoylquinic acid (4,5-DCQA) and kaempferol-3-O-(6″-acetyl)-ß-glucopyranoside (KAG) by MS/MS and (1)H NMR data. Among these compounds, those containing a caffeoyl moiety (5-CQA, FA, 3,5- and 4,5-DCQA) showed relatively strong radical scavenging capacity, with 3,5-DCQA having the greatest radical scavenging capacity in leaf (23.09% of total antioxidant capacity) and root (26.47%) extracts. The relative radical scavenging portion of QAG was only 3.41% in the leaves and KAG did not show any radical scavenging activity. These results demonstrate that the hyphenated HPLC techniques can be successfully applied to rapidly identify structures and evaluate antioxidant activities without prior purification of compounds from plant tissues of P. japonicus.

Study of skin anti-ageing and anti-inflammatory effects of dihydroquercetin, natural triterpenoids, and their synthetic derivatives.

Accessible triterpenoids of ursane and lupane series, the flavonoid dihydroquercetin and their synthetic derivatives with polar substituentss were tested in vitro for inhibition of collagenase 1 (MMP-1) in UVB irradiation assay. Ursolic acid and uvaol disuccinate were the most active inhibitors in the ursane series. In the lupane series, the best inhibition was manifested by carboxymethyl ester of betulonic acid and betulin succinates. Down- regulation of MMP-1 by dihydroquercetin and its synthetic derivatives surpassed the activity of a standard (retinoic acid).

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707.

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

Hepatoprotective effect of flavonoid glycosides from Lespedeza cuneata against oxidative stress induced by tert-butyl hyperoxide.

The aerial parts of Lespedeza cuneata G. Don, perennial legume native to Eastern Asia, have been used therapeutically in traditional Asian medicine to protect the function of liver, kidneys and lungs. However, little is known about the pharmaceutical effect of extracts from this plant. In the present study, the aerial parts of L. cuneata were used to prepare an ethanol extract, which was then tested for hepatoprotective effects against injury by tert-butyl hyperoxide (t-BHP). At a dose of 20 µg/mL, the ethanol extract significantly protected HepG2 cells against the cytotoxicity of t-BHP. Further fractionation of the extract with ethyl acetate allowed the isolation of five flavonoid compounds that were structurally identified by ¹H and ¹³C NMR spectroscopy as isovitexin, hirsutrin, trifolin, avicularin and quercetin. Hirsutrin, avicularin and quercetin (10 µM) showed clear hepatoprotective activity against injury by t-BHP in HepG2 cells, whereas isovitexin and trifolin showed no protective effects. The observed hepatoprotective effect of the investigated compounds showed a high correlation with radical scavenging activity, which followed the structure-activity relationships of the flavonoid aglycones.

Plastoquinones from Sargassum yezoense; chemical structures and effects on the activation of peroxisome proliferator-activated receptor gamma.

Four new plastoquinones, together with two known compounds, sargahydroquinoic acid and sargaquinoic acid, were isolated from the brown alga, Sargassum yezoense collected from the eastern coast of Korea. The structures of these compounds were elucidated based on spectroscopic analyses including NMR and MS. Their structures designated as meroterphenol A (1), B (2), C (3) and D (4) were characterized by a 6-methyl-1,4-benzohydroquinone moiety with an oxygenated diterpenoic acid chain. Meroterphenols A-D showed potent activation effects on peroxisome proliferator-activated receptor gamma (PPARγ).

A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography.

INTRODUCTION: Eisenia bicyclis (Kjellman) Setchell (Laminariaceae) is a common brown alga that inhabits around the coast of Korea, Japan and China. It contains fucoxanthin, a major carotenoid of brown algae which shows a variety of pharmaceutical functions. OBJECTIVE: The aim of this investigation was the quantification and preparative isolation of fucoxanthin from fresh E. bicyclis using a new separation scheme, centrifugal partition chromatography (CPC). METHODOLOGY: The fucoxanthin fraction (Fuco fraction) was prepared by solvent partition method from the acetone extract of fresh E. bicyclis. Fuco fraction was used for CPC using a two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (5:5:7:3, v/v/v/v). The flow rate of mobile phase was 2 mL/min with descending mode while rotating at 1000 rpm. The eluate was monitored at 410 nm. The content and structure of fucoxanthin in the CPC fraction were confirmed with HPLC, UV, APCI/MS and NMR spectra. RESULTS: A preparative CPC yielded 20 mg of fucoxanthin (87% recovery from Fuco fraction) in a two-step separation from 516 mg of Fuco fraction containing 4.59% fucoxanthin. The purity of the isolated fucoxanthin was about 81% in the first CPC step and over 98% in the second CPC step based on the calibration curve. The isolated fucoxanthin was identified as all-trans-fucoxanthin with APCI/MS (parent ion at m/z 641 [M + H - H(2) O](+) ) and (1) H, (13) C and 2-D NMR spectra. CONCLUSION: High-purity fucoxanthin was successfully isolated from fresh E. bicyclis, suggesting further potential applications in the industrial use of this valuable carotenoid.

Tyrosinase inhibition by water and ethanol extracts of a far eastern sea cucumber, Stichopus japonicus.

BACKGROUND: Tyrosinase plays a key role in hyperpigmentaion and enzymatic browning. The present study was aimed at investigating the inhibitory effects of water and 70% aqueous ethanol extracts of Stichopus japonicus, a sea cucumber long consumed as a tonic food and traditional medicine, on the diphenolase activity of tyrosinase. RESULTS: In the tyrosinase inhibition study, high-performance liquid chromatography completely separated L-3,4-dihydroxyphenylalanine and dopachrome from other compounds present in the extracts, and provided more reliable results than the commonly used spectrophotometry. The ethanol extract (IC(50)=0.49-0.61 mg mL(-1)) showed higher inhibitory activity than the water extract (IC(50)=1.80-1.99 mg mL(-1)). Enzyme inhibition by the extracts was reversible and of mixed type. For both extracts, the dissociation constants for binding to free enzyme were significantly smaller than those for binding to enzyme-substrate complex. Ethyl-α-D-glucopyranoside (IC(50)=0.19 mg mL(-1)), isolated for the first time from sea cucumber, and adenosine (IC(50)=0.13 mg mL(-1)), were identified as key tyrosinase inhibitors. CONCLUSION: The sea cucumber extracts were demonstrated to possess considerable inhibitory potency against the diphenolase activity of tyrosinase, suggesting that the sea cucumber may be a good source of safe and effective tyrosinase inhibitors.

Inhibitory effect of ursolic acid derivatives on recombinant human aldose reductase.

Aldose reductase (AR) is the first enzyme in the polyol pathway. AR has been reported to play an important role in the pathogenesis of diabetic complications. Ursolic acid and fourteen synthetic derivatives with ursane skeleton were tested for recombinant human aldose reductase (rhAR) inhibitory activity for development of diabetic complications. Among them, N-(3beta-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) showed most potent rhAR inhibitory activity in vitro. Inhibition mode of N-(3beta-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid (XV) was tested uncompetitively by kinetic analysis using the Lineweaver-Burk plots. Ursolic acid derivative N-(3beta-hydroxyurs-12-en-28-oyl)-4-aminobutyric acid is able to inhibit rhAR uncompetitively and could be offered as a lead compound for AR inhibition.

Anti-inflammatory effects of gomisin N, gomisin J, and schisandrin C isolated from the fruit of Schisandra chinensis.

Schiandra chinensis is a well-known Chinese traditional medicine for the treatment of hepatic disease. In this study, we investigated whether the nine major compounds of Schiandra chinensis could be applied to suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Among the nine lignans, three, gomisin J, gomisin N, and schisandrin C, were found to reduce nitric oxide (NO) production from LPS-stimulated Raw 264.7 cells. These three lignans showed low cytotoxic effects in Raw 264.7 cells. Pre-treatment of Raw 264.7 cells with gomisin J, gomisin N, or schisandrin C reduced the expression of mRNA and the secretion of pro-inflammatory cytokines. These inhibitory effects were found to be caused by blockage of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and c-Jun N-terminal kinase (JNK) phosphorylation.

Preparative separation of chlorogenic acid by centrifugal partition chromatography from highbush blueberry leaves (Vaccinium corymbosum L.).

INTRODUCTION: Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound. OBJECTIVE: The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5-caffeoylquinic acid, 5-CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC). METHODOLOGY: A water fraction containing a high concentration of 5-CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5-CQA from this water fraction using a two-phase solvent system of ethyl acetate-ethanol-water at a volume ratio 4:1:5 (v/v/v). The flow-rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra. RESULTS: The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5-CQA with MS (parent ion at m/z 355.1 [M + H](+)) and (1)H NMR spectra [caffeoyl moiety in the down field (δ 6.0-8.0 ppm) and quinic acid moiety in the up field (δ 2.0-5.5 ppm)]. CONCLUSION: 5-CQA was successfully isolated from blueberry leaves by the CPC method in a one-step procedure, indicating a further potential use for blueberry leaves.

Rapid identification of furanocoumarins in Angelica dahurica using the online LC-MMR-MS and their nitric oxide inhibitory activity in RAW 264.7 cells.

INTRODUCTION: Angelica dahurica (Fisch. Ex hoffm.) Benth. Et Hook. is a perennial herb that grows throughout Korea whose dried roots have been used to treat various diseases in Korean traditional medicine. The root extract contains diverse constituents, and it is necessary to determine the active compounds. OBJECTIVE: To investigate the nitric oxide (NO) inhibitory activity in a root extract of A. dahurica and identify the most active compounds using LC-NMR-MS. METHODOLOGY: In search of the anti-inflammatory constituents of A. dahurica extract, the HPLC-based activity profiling approach was used to investigate the extract's NO inhibitory activity. To directly identify the compounds, a hyphenated LC-NMR-MS technique was applied. Reversed-phase isocratic chromatography was performed using the acetonitrile-water solvent system on a C(30) column. The identification of the compounds was based on information from ESI/MS and 1H-NMR. RESULTS: NO inhibitory activities for five main fractions of the extract were evaluated, which were identified by LC-NMR-MS as containing furanocoumarins: byakangelicol, oxypeucedanin, imperatorin, phellopterin and isoimperatorin. CONCLUSION: The results obtained showed that the anti-inflammatory activities of A. dahurica could be linked to imperatorin and phellopterin.

Effect of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose on elastase and hyaluronidase activities and its type II collagen expression.

In the current era, natural products are gaining prime attention in the fields of cosmeceuticals and pharmaceuticals due to higher safety margins and biological functions, as they have a considerable amount of potential in treating different ailments. Thus, to find effective elastase and hyaluronidase inhibitors from natural resources, fifty Korean plants were screened, and the fruit of Terminalia chebula RETZIUS (Combretaceae) was selected for further structural isolation due to its potent efficacy. The methanol crude extract of the fruits showed 80% elastase and 87% hyaluronidase enzyme inhibition activities at 1 mg/mL. The crude extract, upon bioassay-directed fractionation, led to the isolation of compound 1, whose structure was found by spectral analysis to be 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG). PGG displayed significant elastase and hyaluronidase inhibitory activities with IC50, values of 57 microg/mL and 0.86 mg/mL, respectively; also, treatment of PGG on rabbit articular chondrocytes significantly induced the type II collagen expression. Based on elastase and hyaluronidase inhibitions, and type II collagen expression, it could be suggested that PGG might have an influence on skin conditions when used cosmetically as an active anti-aging ingredient with no cytotoxicity; also, it might be beneficial in relieving painful joint conditions, and thus have relevance for treating arthritis. Therefore, it can be concluded that PGG may prove to be an active ingredient in cosmeceutical and pharmaceutical formulations, and that it definitely merits further in vivo investigations.

Production of lactosucrose from sucrose and lactose by a levansucrase from Zymomonas mobilis.

Lactosucrose (4(G)-beta-D-galactosylsucrose) is an oligosaccharide consisting of galactose, glucose, and fructose. In this study, we prepared lactosucrose from lactose and sucrose using a levansucrase derived from Zymomonas mobilis. Optimum conditions for lactosucrose formation were 23 degrees C, pH 7.0, 18.0% (w/v) lactose monohydrate, and 18% (w/v) sucrose as substrates, and 1 unit of enzyme/ml of reaction mixture. Under these conditions, the lactosucrose conversion efficiency was 28.5%. The product was purified and confirmed to be O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-glucopyranosyl- (1-->2)-beta-D-fructofuranoside, or lactosucrose. A mixed-enzyme system containing a levansucrase and a glucose oxidase was applied in order to increase the efficiency of lactose and sucrose conversion to lactosucrose, which rose to 43.2% as s result.

Preventive and therapeutic effects of blueberry (Vaccinium corymbosum) extract against DSS-induced ulcerative colitis by regulation of antioxidant and inflammatory mediators.

Inflammatory bowel disease (IBD) is an inflammatory disorder caused by hyperactivation of effector immune cells that produce high levels of proinflammatory cytokines. The aims of our study were to determine whether orally administered blueberry extract (BE) could attenuate or prevent the development of experimental colitis in mice and to elucidate the mechanism of action. Female Balb/C mice (n=7) were randomized into groups differing in treatment conditions (prevention and treatment) and dose of BE (50 mg/kg body weight). Acute ulcerative colitis was induced by oral administration of 3% dextran sodium sulfate for 7 days in drinking water. Colonic mucosal injury was assessed by clinical, macroscopic, biochemical and histopathological examinations. BE significantly decreased disease activity index and improved the macroscopic and histological score of colons when compared to the colitis group (P<.05). BE markedly attenuated myeloperoxidase accumulation (colitis group 54.97±2.78 nmol/mg, treatment group 30.78±1.33 nmol/mg) and malondialdehyde in colon and prostaglandin E2 level in serum while increasing the levels of superoxide dismutase and catalase (colitis group 11.94±1.16 U/ml, BE treatment group 16.49±0.39 U/ml) compared with the colitis group (P<.05). mRNA levels of the cyclooxygenase (COX)-2, interferon-γ, interleukin (IL)-1ß and inducible nitric oxide synthase cytokines were determined by reverse transcriptase polymerase chain reaction. Immunohistochemical analysis showed that BE attenuates the expression of COX-2 and IL-1ß in colonic tissue. Moreover, BE reduced the nuclear translocation of nuclear transcription factor kappa B (NF-κB) by immunofluorescence analysis. Thus, the anti-inflammatory effect of BE at colorectal sites is a result of a number of mechanisms: antioxidation, down-regulation of the expression of inflammatory mediators and inhibition of the nuclear translocation of NF-κB.

Optimisation of pressurised liquid extraction of antioxidants from black bamboo leaves.

To develop an efficient green extraction approach for recovering bioactive compounds from natural plants, the potential of using pressurised liquid extraction (PLE) was examined on black bamboo (Phyllostachys nigra) leaves, with ethanol/water as solvents. The superheated PLE process showed a higher recovery of most constituents and antioxidative activity, compared to reflux extraction, with a significantly improved recovery of the total phenolic (TP) and flavonoid (TF) content and DPPH radical scavenging ability. For a broad range of ethanol aqueous solutions and temperatures, 50% EtOH and 200°C (static time: 25min) gave the best performance, in terms of the TP and TF (75% EtOH) content yield and DPPH scavenging ability (25% EtOH). Under the optimised extraction conditions, eight main antioxidative compounds were isolated and identified with HPLC-ABTS(+) assay guidance and assessed for radical scavenging activity. The superheated extraction process for black bamboo leaves enhanced the antioxidant properties by increasing the extraction of the phenolic components.