ABSTRACT
Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 µg/d) has been recommended for adults in the European Union, and its increased intake (600 µg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).
Subject(s)
Folic Acid Deficiency , Folic Acid , Adult , Animals , Female , Flour , Folic Acid/metabolism , Folic Acid Deficiency/prevention & control , Food, Fortified , Humans , Infant, Newborn , Mammals/metabolism , Pregnancy , Triticum/metabolismABSTRACT
AIMS: Noninvasive music adjuvant therapy shows great potential in improving seizure control when combined with routine antiepileptic drugs. However, the diversity of previous music protocols has resulted in disparate outcomes. The optimized protocol and features for music adjuvant therapy are still not fully understood which limits its feasibility. METHODS: By applying different regimens of music therapy in various temporal lobe epilepsy models, we evaluated the effect of music in combination with sub-dose drugs on epileptic seizures to determine the optimized protocol. RESULTS: A subgroup of kindled mice that were responsive to music adjuvant therapy was screened. In those mice, sub-dose drugs which were noneffective on kindled seizures, alleviated seizure severity after 12 h/day Mozart K.448 for 14 days. Shorter durations of music therapy (2 and 6 h/day) were ineffective. Furthermore, only full-length Mozart K.448, not its episodes or other music varieties, was capable of enhancing the efficacy of sub-dose drugs. This music therapeutic effect was not due to increasing cerebral drug concentration, but instead was related with the modulation of seizure electroencephalogram (EEG) spectral powers in the hippocampus. CONCLUSION: These results indicate that long-term full-length Mozart K.448 could enhance the anti-seizure efficacy of sub-dose drugs and may be a promising noninvasive adjuvant therapy for temporal lobe epilepsy.
Subject(s)
Anticonvulsants/pharmacology , Epilepsy, Temporal Lobe/therapy , Music Therapy , Animals , Anticonvulsants/administration & dosage , Combined Modality Therapy , Disease Models, Animal , Electroencephalography , Epilepsy, Temporal Lobe/drug therapy , Male , Mice , Mice, Inbred C57BL , Time Factors , Valproic Acid/pharmacologyABSTRACT
Fupo Ganmao Granules (FP) is a classic traditional Chinese medicine (TCM) formula, composed of 4 herbal medicines, Folium Hibisci Mutabilis, Magnolia officinalis, Pericarpium Citri Reticulatae, and Fructus arctii. It is regularly used in the treatment of common cold with wind-heat syndrome. The biologically active constituents in FP, especially those in trace amount, remain unclear. In this study, the components of FP were profiled and characterized using HPLC-ESI-MS2. A total of 58 compounds were identified according to their fragmentation features in IT-TOF-MS and IT-MS2, including 13 phenolic acids, 17 flavonoids, 21 lignans, 2 alkaloids, and 5 other compounds. Among them, the identities of 8 compounds were explicitly confirmed by comparing them with standard compounds, and their contents were determined. Our study provided a comprehensive understanding of the diverse chemical profile in FP, and the approach we developed is useful for the analysis of other TCM formulas.
Subject(s)
Chemistry, Pharmaceutical/methods , Drugs, Chinese Herbal/analysis , Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Tandem Mass Spectrometry/methodsABSTRACT
RATIONALE: Two structurally similar bioactive regioisomers, 3-O-tetradecanoylingenol and 20-O-tetradecanoylingenol, from Euphorbia wallichii presented quite different fragmentation behaviors. Revealing the related fragmentation pathways may improve the efficiency of characterization and identification of such type of compounds. METHODS: The two regioisomers were subjected to collision-induced dissociation (CID) on Finnigan LCQ(DECA) and LTQ Orbitrap XL instruments. Based on the CID results, the possible fragmentation pathways were proposed and investigated with density functional theory (DFT) calculations. RESULTS: Elimination of C(14)H(28)O(2) (tetradecanoic acid) for 3-O-tetradecanoylingenol and the sequential losses of C(4)H(8) (butylene) for 20-O-tetradecanoylingenol were observed in ESI-MS/MS, witnessed also by HR-ESI-MS/MS. The fragmentation pathways were proposed and verified by calculating the activation energy of their transition states by DFT calculations. CONCLUSIONS: Based on the observations, fragmentation pathways for the two regioisomers were proposed and verified by calculating the energy of the reactants, products and the corresponding transition states using DFT. This report should have value in rapid identification of the derivatives of ingenol and other regioisomers in natural products.
Subject(s)
Diterpenes/chemistry , Euphorbia/chemistry , Plant Extracts/chemistry , Tandem Mass Spectrometry/methods , Models, MolecularABSTRACT
Two previously unknown resveratrol trimers named wilsonols A-B, as well as a resveratrol tetramer named wilsonol C, were isolated from Vitis wilsonae Veitch, together with 12 known oligostilbenes. Their chemical structures have been elucidated by detailed analyses of 1D and 2D NMR spectroscopic data, as well as chemical evidence obtained via either catalysis with HRP (horseradish peroxidase) and H(2)O(2) (hydrogen peroxide), acid, or UV irradiation. During the chemical processes, a biomimetic resveratrol tetramer named diviniferin B that has not been found in nature was obtained. These oligostilbenes showed potent scavenging abilities towards DPPH radicals and selective quenching effects on (1)O(2) radicals. Furthermore, the biogenetic transformations between the 16 oligostilbenes have been elaborated chemically to provide a comprehensive mechanism of the antioxidative defense system in this plant species.
Subject(s)
Free Radical Scavengers/chemistry , Oxygen/chemistry , Stilbenes/chemistry , Vitis/chemistry , Biphenyl Compounds/chemistry , Free Radical Scavengers/isolation & purification , Picrates/chemistry , Plant Extracts/chemistry , Resveratrol , Stilbenes/isolation & purificationABSTRACT
The polar compounds such as alkaloid compounds are important bioactive components in traditional Chinese medicines. In present study, a comprehensive method for separation and analysis of polar compounds from the polar fraction of traditional Chinese medicine Stephania yunnanensis was established. Both the major components and minor components were analyzed by counter-current chromatography combined with liquid chromatography tandem mass spectrometry (LC-MS(n)). From 50 mg polar fraction of crude extract, 15.2mg corydine and 4.8 mg stepharine with purities over 90% were successfully separated via a polar solvent system n-butanol: methanol: water (4:1:5, v/v) with 10 mM NaOH as an additive in the lower phase, in one step operation. Their structures were further identified by 1H NMR and FTICR-MS. Besides, three minor components were identified by HPLC-MS(n) based on the fragmentation behavior of the purified compounds.
Subject(s)
Alkaloids/isolation & purification , Chromatography, High Pressure Liquid/methods , Countercurrent Distribution/methods , Stephania/chemistry , Tandem Mass Spectrometry/methods , 1-Butanol , Alkaloids/analysis , Drugs, Chinese Herbal/chemistry , Methanol , Sodium Hydroxide , WaterABSTRACT
A rapid and stable method consisting of high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for the identification and differentiation of common diastereoisomeric ursane-type triterpenoids at the C-3 position. Two characteristic fragment ions, [M-H-H(2) O-CO(2)](-) and [M-H-H(2)O-HCOOH](-) , exhibited significant stereochemical effects and were utilized to distinguish 3-OH epimers. Based on reference standards, the abundance of the fragment ion [M-H-H(2)O-HCOOH](-) in 3ß-OH compounds in the MS(3) experiment was dramatically higher compared to [M-H-H(2) O-CO(2)](-); however, for 3α-OH compounds, the product ion [M-H-H(2) O-CO(2)](-) was noted to be higher than [M-H-H(2)O-HCOOH](-). Energy-resolved mass spectrometric experiments were carried out to support the differentiation of these diastereoisomeric triterpenoids at the C-3 position. Using this method, a total of nine ursane-type triterpenoids from a plant crude extract, including four pairs of epimers at the C-3 position, were identified and distinguished rapidly. Furthermore, offline Fourier transform ion cyclotron resonance tandem mass spectrometry was also performed to assign accurate elemental compositions.
Subject(s)
Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Triterpenes/isolation & purification , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Rosaceae/chemistry , Stereoisomerism , Triterpenes/chemistryABSTRACT
An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.
Subject(s)
Anthraquinones/isolation & purification , Countercurrent Distribution/methods , Drugs, Chinese Herbal/isolation & purification , Rheum/chemistry , Anthraquinones/chemistry , Countercurrent Distribution/instrumentation , Molecular StructureABSTRACT
The hepatoprotective and antioxidant activities of the n-butanol extract of Rubus parvifolius L. (RPL), a widely used medicinal plant, were evaluated. Results demonstrated that RPL extract possessed pronounced hepatoprotective effects against carbon tetrachloride (CCl(4))-induced hepatic injury in mice, which was at least partially attributed to its strong antioxidant capacity. Treatment with RPL extract markedly attenuated the increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels caused by CCl(4) intoxication. It also significantly prevented the decrease in superoxide dismutase (SOD) activity and the increase in malondialdehyde (MDA) content of liver tissue. Meanwhile, histopathological changes of hepatic damage were also remarkably ameliorated. Phytochemical analysis based on high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) revealed the presence of various phenolic compounds, including caffeic acid conjugates, ellagic acid glycosides, and flavonol glycosides, which might be responsible for the hepatoprotective and antioxidant activities of RPL.
Subject(s)
Antioxidants/pharmacology , Liver/drug effects , Plants, Medicinal , Rosaceae , 1-Butanol , Alanine Transaminase/blood , Animals , Antioxidants/chemistry , Aspartate Aminotransferases/blood , Carbon Tetrachloride/toxicity , Chromatography, High Pressure Liquid , Drug Evaluation, Preclinical , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Female , Free Radical Scavengers/pharmacology , Liver/injuries , Liver/metabolism , Liver/pathology , Male , Malondialdehyde/metabolism , Mice , Plants, Medicinal/chemistry , Rosaceae/chemistry , Superoxide Dismutase/metabolism , Tandem Mass SpectrometryABSTRACT
A practical approach for rapidly screening antioxidants against superoxide anion radicals from complex mixtures was developed based on the quantitative difference in active compounds before and after their reaction. To test the effectiveness of the approach, seven flavonoids with antioxidative properties were investigated both individually and in a mixture. Using the approach, antioxidants could be rapidly separated and screened with a ranked order of activities in the meantime. The radical scavenging activities were in the following order: quercetin > kaempferol > fisetin > puerarin > luteolin > rutin > baicalein. The order of activity was conducted by comparing the scavenging ratio of the antioxidant, which was completely consistent with the results obtained from the traditional electronic spin resonance. Then, the method was successfully applied to black tea extracts. This approach is fast and convenient for screening, isolating, and analyzing potential antioxidants from a mixture with good quantitative and reproducible ability.
Subject(s)
Antioxidants/analysis , Antioxidants/chemistry , Complex Mixtures/chemistry , Free Radical Scavengers/chemistry , Superoxides/chemistry , Anions/chemistry , Chromatography, High Pressure Liquid , Plant Extracts/chemistry , Reproducibility of Results , StereoisomerismABSTRACT
Dracorhodin is a major constituent found in "Dragon's blood" resin of Daemonorops draco Willd. Blume. This natural flavylium compound is a potent pharmaceutical substance due to its biological and pharmacological activities such as antimicrobial, antiviral, antitumor and cytotoxic activity. An effective high-speed counter-current chromatography method was successfully established for the isolation and purification of dracorhodin directly from extract of D. draco by using a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (2:3:2:3 v/v). Under the optimal conditions, 6.6 mg dracorhodin was obtained from 100 mg crude resin. The isolated fraction of counter-current chromatography was determined by HPLC, NMR, UV/visible and ESI/MS combined with pH modulation, since dracorhodin is unstable in solution which exists in different forms depending on pH values. The data were compared with those of the reference substance, and the literatures as well. The purity of dracorhodin was over 98% based on the HPLC result.
Subject(s)
Arecaceae/chemistry , Benzopyrans/isolation & purification , Countercurrent Distribution/methods , Drugs, Chinese Herbal/chemistry , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Plants, Medicinal/chemistry , Resins, Plant/chemistry , Solvents , Spectrometry, Mass, Electrospray Ionization , SpectrophotometryABSTRACT
Euonymus hederaceus is distributed widely in the south of China; its stems and leaves have been used as folk medicines to treat many diseases such as renal deficiency and chronic diarrhea, traumatic injury, and abnormal menstruation. Chemical investigation of the leaves and stems of Euonymus hederaceus resulted in the isolation forthe first time and characterization of a new friedelane type triterpene with a molecular mass of 472 and molecular formula of C(30)H(48)O(4)by high resolution mass spectrometry. The (1)H-NMR (13)C-NMR and DEPT135(0)spectra matched the characteristic data of the proposed triterpene skeleton.The compound was finally identified as 28-hydroxyfriedelan-3-one-29-oic acid on the basis of spectroscopic evidence, including two dimensional nuclear magnetic resonance as well as its IR spectrum.
Subject(s)
Euonymus/chemistry , Triterpenes/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Spectrophotometry, Infrared , Triterpenes/isolation & purificationABSTRACT
High-performance liquid chromatography/electrospray tandem mass spectrometry was developed to distinguish isomers and compounds of similar structures with different configurations in the rhizomes of Z. cassumunar. Energy-resolved breakdown curves were utilized to differentiate four compounds. Compounds 2 (3R,4S) and 4 (3R,4R) were a pair of stereoisomers which could be distinguished easily by breakdown curves. The breakdown curve of compound 1 was identical to that of compound 2, which suggested that the configuration of compound 1 was (3R,4S) or (3S,4R). The breakdown curve of compound 3 was completely different from those of compounds 1, 2 and 4, and it might be that the configuration of the double bond of compound 3 was different from the other three compounds. Hence, the described method using breakdown curves has great potential in the distinguishing of isomers and compounds of similar structure with different configurations.
Subject(s)
Biphenyl Compounds/chemistry , Stilbenes/chemistry , Tandem Mass Spectrometry/methods , Zingiberaceae/chemistry , Chromatography, High Pressure Liquid , Dimerization , Plant Extracts/chemistry , StereoisomerismABSTRACT
High-speed counter-current chromatography (HSCCC) was applied to the preparative isolation and purification of two amides from Mallotus lianus Croiz. In a single HSCCC separation, using the two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (5:1:5:1 v/v), 247.5 mg of the enriched crude sample was separated to afford 10.3 mg of N-isobutyl-2E,4E,12Z-octadecatrienamide and 15.7 mg of (7Z,10Z,18Z)tricosa-7,10,18-trienamide, a novel compound, with the purities of 98.0 and 94.6%, respectively. The HSCCC fractions were analyzed by HPLC and chemical structures of the compounds were identified by 1D- and 2D-NMR, ESI-, and GC-MS.
Subject(s)
Amides/isolation & purification , Countercurrent Distribution/methods , Euphorbiaceae/chemistry , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Extracts/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, UltravioletABSTRACT
Five trace impurities were detected in the bulk drug citalopram using high-performance liquid chromatography with UV detection. A simple and sensitive method suitable for liquid chromatography tandem multistage mass spectrometry (HPLC/MS(n)) analysis was developed. Using this method, the fragmentation behavior of citalopram and the impurities was investigated. Four impurities were rapidly characterized, and an unknown impurity was elucidated as 3-(3-dimethylaminopropyl)-N-(1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)-3-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide on the basis of the MS(n) and exact mass evidence, and the proposed structure was further confirmed by nuclear magnetic resonance (NMR) experiments after preparative isolation.
Subject(s)
Chromatography, High Pressure Liquid/methods , Citalopram/analysis , Citalopram/chemistry , Drug Contamination/prevention & control , Drug Evaluation, Preclinical/methods , Spectrometry, Mass, Electrospray Ionization/methods , Spectroscopy, Fourier Transform Infrared/methods , Antidepressive Agents, Second-Generation/analysis , Antidepressive Agents, Second-Generation/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The medicinal plant Rubia cordifolia has been used widely in traditional Chinese medicine (TCM) for its antibacterial, antioxidant and anti-inflammatory activities. In this study, a preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of the bioactive component mollugin directly from the ethanol extract of R. cordifolia was successfully established by using light petroleum (bp 60-90 degrees C)/ethanol/diethyl ether/water as the two-phase solvent system. The upper phase of light petroleum/ethanol/diethyl ether/water (5:4:3:1 v/v) was used as the stationary phase of HSCCC. Under the optimum conditions, 46 mg of mollugin at 98.5% purity, as determined by HPLC, could be yielded from 500 mg of the crude extract in a single HSCCC separation. The peak fraction of HSCCC was identified by 1H NMR and 13C NMR.
Subject(s)
Drugs, Chinese Herbal/isolation & purification , Ethanol , Pyrans/isolation & purification , Rubia/chemistry , Chromatography, High Pressure Liquid/methods , Countercurrent Distribution/methods , Drugs, Chinese Herbal/chemistry , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Pyrans/chemistryABSTRACT
A reversed-phase liquid chromatography/tandem multistage mass spectrometry (MS/MS) method was developed for the characterization of amides from the extracts of Piper longum. The characteristic fragmentations of the amides found in P. longum showed diagnostic structural information. Extracted ion chromatography (EIC) and constant neutral losses were used to guide the search for the amides of interest. Amides of known structures that contain four subtypes of amides were rapidly determined, and novel amides were also identified for this plant. Forty-two amides were rapidly identified, of which 22 were found in this plant for the first time and 9 were new compounds. The method is convenient and sensitive, especially for minor components in the unpurified, complex mixture; the structures of unknown constituents could be determined, in the absence of authentic sample, by comparison of the fragmentation patterns with those of homologous compounds.
Subject(s)
Amides/chemistry , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal/chemistry , Piper/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Amides/metabolism , Drugs, Chinese Herbal/metabolism , Piper/metabolism , Plant Extracts/chemistry , Plant Extracts/metabolismABSTRACT
A preparative two-dimensional counter-current chromatographic system (2D-CCC) for simultaneous separation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens was presented. It was based on the use of a high-speed CCC (HSCCC, total column volume V(c)=146ml) instrument in the first dimension (1st-D) and a preparative upright CCC (UCCC, V(c)=1500ml) column in the second dimension (2nd-D). The interface was a homemade column-switching system with a 50-ml sample loop. In this way, almost the whole interested region from the first dimension was transferred on-line to the second dimension for further separation. The use of a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5 and 3:5:3:5, v/v) in the two dimensions permitted the simultaneous separation of oridonin and ponicidin, making this 2D-CCC system with satisfactory resolution and peak capacity. During about 9h separation, two injections with about a 250mg amount of the crude extract for each, was accomplished using this 2D-CCC system, yielding 60mg of oridonin (1) and 10mg of ponicidin (2) all at a purity of over 95% as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization (ESI) MS, (1)H NMR and (13)C NMR.
Subject(s)
Countercurrent Distribution/methods , Diterpenes, Kaurane/analysis , Diterpenes/analysis , Isodon/chemistry , Chromatography, High Pressure Liquid , Countercurrent Distribution/instrumentation , Diterpenes/chemistry , Diterpenes/isolation & purification , Diterpenes, Kaurane/chemistry , Diterpenes, Kaurane/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Extracts/analysis , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Reproducibility of Results , Spectrometry, Mass, Electrospray IonizationABSTRACT
An alkali-extractable polysaccharide (1) was isolated from the seeds of Retama raetam ssp. gussonei. Its composition and linkage determination have been investigated using component analysis, methylation analysis, hydrolysis studies, and NMR spectroscopic analysis. This was shown to contain d-xylose, d-glucose, and 4-O-methyl-d-glucuronic acid in a molar ratio of 7:2:1 and consisted of a backbone of beta-(1-->4)-linked d-xylopyranosyl residues, having branches of alpha-d-glucopyranosyl residues that contain (1-->2)-linkages and 4-O-methyl-alpha-d-glucopyranosyluronic acid at O-2. This is the first report on the isolation of an alkali-extractable polysaccharide from this plant.
Subject(s)
Fabaceae/chemistry , Plants, Medicinal/chemistry , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Endo-1,4-beta Xylanases/metabolism , Seeds/chemistry , Trichoderma/enzymologyABSTRACT
In this study, the edible mycelia of Cordyceps sinensis (Berk.) Sacc. were sequentially extracted by petroleum ether, ethyl acetate, ethanol and ultrasonic water-bath (75 degrees C), and the water extract was further isolated by Sephadex G-100 to afford a petroleum ether extract (PE), ethyl acetate extract (EAE), ethanol extract (EE), glycoprotein (GP) and a purified polysaccharide (PS). In combination with component analysis, the isolated PS showed d-Glc, d-Man, l-Ara and d-Gal in a molar ratio of 8:90:1:1. The average molecular weight of PS was determined as approximately 8.3 x 10(4). The immunomodulatory potentials of these samples (PE, EAE, EE, GP and PS) at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were studied. ICR mice were immunized subcutaneously with 100 microg OVA alone or with 100 microg OVA dissolved in saline containing these samples (100, 200 or 400 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)- and OVA-stimulated splenocyte proliferation and OVA-specific antibody in serum were investigated. These samples, except for PS, significantly enhanced the Con A- and OVA-induced splenocyte proliferation in OVA-immunized mice at a suitable dose (p < 0.05 or p < 0.01). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were significantly enhanced by these extracts and PS compared with the OVA control group (p < 0.05, p < 0.01 or p < 0.001).