Process development for mycelial growth and polysaccharide production in Tricholoma matsutake liquid culture

In this study, the effects of agitation and aeration on mycelial growth and exo-polysaccharide production were examined in batch cultures of Tricholoma matsutake. Agitation was varied from 100 to 300 rpm and aeration was varied from 0.5 to 1.5 vvm. Mycelial growth was 21.87 g/l at 150 rpm, and exo-polysaccharide production was 8.79 g/l at 1.5 vvm. When we analyzed the polysaccharide extractions from the cultured mycelium and the culture broth of T. matsutake, 1.4 g of crude polysaccharide was found per 100 g of dried weight in the cultured mycelium, and 1.47 g/l of polysaccharides was found in the culture broth. In addition, the amounts of β-Glucan in the soluble polysaccharide fractions of the cultured mycelium and culture broth were 75.4% and 83.6%, respectively. The cultured mycelium and the culture broth contained a higher amount of β-Glucan than that of the fruiting body.     

Structural characterisation and antioxidant properties of polysaccharides from the fruiting bodies of Russula virescens

Water-soluble crude polysaccharide named as RVP was obtained from the fresh fruiting bodies of Russula virescens by boiling-water extraction. DEAE-Sepharose CL-6B column chromatography was used for the fractionation of polysaccharide RVP. Two fractions were obtained, namely RVP-1 and RVP-2. RVP-1 and RVP-2 were composed mainly of glucose, with the estimated equivalent dextran molecular weights of 3.1 × 10⁵ and 4.2 × 10⁵ Da, respectively. Analysis by Periodate oxidation–Smith degradation indicated that RVP-1 was composed of 68.3% (1 →)- or ((1 → 6)-glycosidic linkages and 31.7% (1 → 3)-glycosidic linkages, and RVP-2 7.9% (1 →)- or (1 → 6)-glycosidic linkages, 9.6% (1 → 2)-glycosidic linkages, 35.7% (1 → 4)-glycosidic linkages, and 46.8% (1 → 3)-glycosidic linkages. RVP-1 exhibited equivalent inhibiting power for self-oxidation of 1,2,3-phentriol to vitamin C (Vc), a little higher scavenging activity of superoxide radical and hydroxyl radical than Vc. The reducing power of RVP-1 at 20.0 mg/ml was 0.77, and RVP-1 was a good chelating agent for ferrous ions. Overall, RVP-1 possessed good antioxidant properties and should be developed as a novel potential antioxidant.     

Purification,preliminary structural characterization and in vitro antioxidant activity of polysaccharides from Acanthus ilicifolius

Crude Acanthus ilicifolius (A. ilicifolius) polysaccharides (CAIP) were obtained by hot water extraction and deproteinated. Two major polysaccharide fractions, the neutral A. ilicifolius polysaccharide (NAIP) and the acid A. ilicifolius polysaccharide B (AAIP-B), were isolated from CAIP by chromatography using DEAE-Sepharose Fast Flow and Sepharose CL-6B. The molecular weights (M_w) of NAIP and AAIP-B, determined by high performance gel-filtration chromatography (HPGFC), were 11,775 and 23,161 Da, respectively. AAIP-B contained 51.23% uronic acid, characteristic of a pectin-type hetero-polysaccharide. Analysis of the neutral monosaccharide composition indicated that NAIP contained high proportions of arabinose, galactose and glucose. However, AAIP-B was mainly composed of rhamnose, arabinose and galactose. Their structure properties were confirmed by FT-IR.     

Free radical-scavenging and inhibition of nitric oxide production by four grades of pine mushroom (Tricholoma matsutake Sing.)

Fractions from methanol extracts of four grades of pine mushroom (Tricholoma matsutake Sing.) were evaluated for their free radical-scavenging and inhibition of nitric oxide production, and the underlying mechanisms were elucidated. The fractions from first-grade pine mushroom exhibited the highest degree of free radical-scavenging and inhibition of nitric oxide production among the various grades of mushroom tested, and free radical-scavenging and inhibition of nitric oxide production by the second-, third-, and fourth-grade mushroom fractions were successively lower. The degree of free radical-scavenging by each fraction decreased in the order ethyl acetate > butanol > diethyl ether > water. Inhibition of nitric oxide production by each fraction decreased in the order ethyl acetate > butanol > water. The ethyl acetate and butanol fractions of methanol-extracted pine mushroom samples exhibited potential anti-oxidative and anti-inflammatory effects that might be attributable to phenolics or flavonoids.     

Structural characterisation of acid- and alkali-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their immunomodulatory activities

The acid- and alkali-soluble polysaccharides (DIPs I and II) were extracted from the fruiting body of Dictyophora indusiata in this work. DIP I comprised of Glc (glucose), Fru (fructose) and Man (mannose), whereas DIP II was of Glc and Fru. Glc was the dominant monosaccharide in both DIPs with relatively molar percentage of >60%, which constructed the backbone of polysaccharide chain. The analysis of the glycosidic linkages indicated that Glc occurred as Glc 1 → 6 in DIP II. The immunological assays showed that both DIPs I and II had a noticeable effect on the hemolysis antibody level in the tested dosage range. However, DIP I could improve the weight of thymus organ and phagocytosis of monocyte. DIP II could restore delayed-type hypersensitivity reaction to dinitrofluorobenzene (DNFB), improving the activity of natural killer cells and the proliferation of splenocytes at high dose.     

Phenolics from hull of Garcinia mangostana fruit and their antioxidant activities

The air-dried fruit hulls of Garcinia mangostana Linn. were extracted with 70% MeOH, and then partitioned into the n-BuOH fractions. Furthermore, three major phenolic components related to their antioxidant activities were purified by silica gel column chromatography and Sephadex LH-20 and then identified as P₁ (1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl)), P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin) using UV–visible spectrophotometry, IR spectrophotometry and NMR spectroscopy, respectively. The antioxidant activities of three major phenolics were evaluated using different tests, including the free radical scavenging capability and total antioxidant activity in a linoleic acid peroxidation. These three phenolic compounds exhibited different antioxidant activities in these antioxidant tests. The hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capabilities and the activity against linoleic acid peroxidation of P₁ were greater than those of P₂ and P₃, while the superoxide anion radical scavenging activity of P₃ was greater than that of P₁, but was close to that of P₂ or α-tocopherol. An activity-guided isolation and purification process was used to identify the components showing the strong DPPH radical scavenging activity from G. mangostana Linn.     

Three sulphated polysaccharides isolated from the mucilage of mud snail, Bullacta exarata philippi: Characterization and antitumour activity

Three sulphated polysaccharides, coded as BEMPA, BEMPB₁, BEMPB₂, were extracted from the mucilage of mud snail of Bullacta exarata and purified by DEAE-cellulose ion-exchange and size-exclusion chromatography. Structural analysis of purified polysaccharides by chemical and biochemical methods revealed BEMPA was a high (1→3,4)-linked mannose-containing polysaccharide with molecular weight of 22,977 Da. BEMPB₁, with molecular weight of 64,117 Da, was a high (1→3)-linked arabinose-containing polysaccharide. BEMPB₂ was mainly composed of (1→3,4)-linked mannose with molecular weight of 47,507 Da. The comparison between sulphated polysaccharides and their desulphated products showed that sulphate substitutions of BEMPB₁ were deduced to be at the C-3 of (1→4)-linked mannose, while sulphate substitutions of BEMPA and BEMPB₂ were at C-4 of (1→3)-linked mannose. Furthermore, BEMPA exhibited highest inhibitory effects on growth of B-16 melanoma cells, and IC₅₀ were 31.1 μg/mL.     

Comparison of umami-taste active components in the pileus and stipe of pine-mushrooms (Tricholoma matsutake Sing.) of different grades

Umami-taste active components in pine-mushrooms (Tricholoma matsutake Sing.) were compared according to different grades (first, second, third, and fourth) and parts (pileus and stipe). The contents of umami-taste active free amino acids, such as aspartic acid and glutamic acid, decreased in the order of second grade > third grade > fourth grade > first grade. Also, the contents of umami-taste active 5′-nucleotides were highest and lowest in pine-mushrooms of second and first grades, respectively. All the contents of umami-taste active amino acids and 5′-nucleotides were higher in the pileus than in the stipe, irrespective of pine-mushroom grades. Equivalent umami concentration values of pine-mushrooms ranged from 13.26 (in the stipe of first grade) to 204.26 g (in the pileus of second grade) per 100 g. All the quantitative data obtained in this study are highly consistent with our previously data on umami sensory intensities of pine-mushrooms.     

Purification, antitumour and immunomodulatory activity of water-extractable and alkali-extractable polysaccharides from Solanum nigrum L.

The water-extractable and the alkali-extractable polysaccharides from Solanum nigrum L. (named SNLWP and SNLAP, respectively) and their four polysaccharide sub-fractions (SNLWP-1, SNLWP-2, SNLAP-1 and SNLAP-2), were isolated and purified by ethanol precipitation, dialysis, anion-exchange and gel filtration chromatography. Antitumour and immunomodulatory activity of the polysaccharides was evaluated by determining the survival time, the ascites volume, the weight of immune organ and the level of cytokine (IL-2, IL-10 and IFN-γ) of H₂₂-bearing mice, respectively. The results showed that SNLWP-1, SNLAP-1 and SNLAP-2 had significant antitumour and immunomodulatory activity, whereas SNLWP-2 hardly demonstrated the activity. SNLWP-1 contained galactose and arabinose as main sugar components, and both SNLAP-1 and SNLAP-2 were rich in xylose, galactose and arabinose. SNLWP-2 was rich in glucose. In conclusion, SNLWP-1, SNLAP-1 and SNLAP-2 have potent antitumour activity which may be associated with their potent immunostimulating effect and monosaccharide composition.     

Polyphenols and triterpenes from Chaenomeles fruits: Chemical analysis and antioxidant activities assessment

Mugua, fruit of the genus Chaenomeles, is a valuable source of health food and Chinese medicine. To elucidate the bioactive compounds of five wild Chaenomeles species, extracts from fresh fruits were investigated by HPLC-DAD/ESI-MS/MS. Among the 24 polyphenol compounds obtained, 20 were flavan-3-ols (including catechin, epicatechin and procyanidin oligomers). The mean polymerisation degree (mDP) of procyanidins was examined by two acid-catalysed cleavage reactions; the mDP value was the highest in Chaenomeles sinensis and the lowest in Chaenomeles japonica. Total polyphenol content (TPC) reached 46.92 and 46.28 mg/g gallic acid equivalents (GAE) in Chaenomeles speciosa and Chaenomeles thibetica, respectively, although their main bioactive compounds were different (being epicatechin and procyanidin B2 in the former, and catechin and procyanidin B1 in the latter). These two species also exhibited equally strong free radical scavenging activities. Our results further showed that the antioxidant ability of Chaenomeles fruits was significantly correlated to their total polyphenol contents. Two triterpenes (oleanolic acid and ursolic acid) were the highest quantity in Chaenomeles cathayensis and C. thibetica, respectively.     

Characterisation of esterolytic activity from two wild mushroom species, Amanita vaginata var. vaginata and Tricholoma terreum

Characterisation of esterase activities from the edible mushroom species, Amanita vaginata var. vaginata and Tricholoma terreum, were investigated. Native electrophoresis of the crude extracts prepared from both mushroom samples showed the presence of esterolytic activities. The extracts had the greatest activity in the presence of p-nitrophenyl butyrate (pNPB) as a substrate. pH and temperature optima were found to be 8.0 and 30 °C for both enzymes, respectively. V_max and K_m values were determined as 14.2 U/l and 71 μM for A. vaginata var. vaginata and 34.6 U/l and 9.6 μM for T. terreum, respectively. The pH-stability profile showed a stationary line between 3.0 and 10.0 for both enzymes. The esterolytic activities from the extracts were maintained between 10 and 40 °C for 4 h and started to decrease at 50 °C. The effects of EDTA, NaN₃, DTT and PMSF on the enzyme activity were also investigated.     

Preparation,identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa

Ultrafiltration membranes of different pore size were applied to fractionate Chlorella pyrenoidosa polysaccharides (CPPS) and the main fraction could be separated by a membrane with nominal molecular weight cut-off (NMWCO) of 30 kDa. Ultrafiltration parameters of 40 °C 14.0 psi were optimized for obtaining the main fraction. The resulting sample was further purified by anion-exchange chromatography and size exclusion chromatography, and two distinctive polysaccharides, CPPS Ia and IIa were recovered. CPPS IIa had infrared spectral characteristic of polysaccharides similar to CPPS Ia, and the symmetrical stretching peak at 1408–1382 cm⁻¹ was an indication of the presence of carboxyl groups. The peak molecular weights were 69658 Da and 109406 Da, for CPPS Ia and CPPS IIa, respectively. Both CPPS Ia and IIa were composed of rhamnose, mannose, glucose, galactose and an unknown monosaccharide. Galactose (relative mass 46.5%) was the predominant monosaccharide of CPPS Ia and in CPPS IIa, rhamnose (37.8%) was predominant. CPPS Ia and IIa presented significantly higher antitumor activity against A549 in vitro than did a blank control, in a dose-dependent manner. Both fractions might be useful for developing natural safe antitumor drugs from C. pyrenoidosa resources.     

Biological activities of flavonoids from Nitraria retusa (Forssk.) Asch. and their acylated derivatives

The antioxidant activity and antiproliferative property towards Caco2 cells of water and methanol extracts/fractions of Nitraria retusa were investigated. The total phenolic and flavonoid contents of the extracts/fractions were determined, and the four major flavonoids were identified as isorhamnetin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-robinobioside. The results showed a relationship between the extracts/fractions activities and their flavonoid contents. Moreover, the chloroform extract which was enriched with the aglycone flavonoid isorhamnetin exhibited the highest activities. The activities of N. retusa flavonoids were compared to those of model flavonoids, quercetin, isoquercitrin and rutin. In all cases, the aglycone compounds were more active than their glycosylated derivatives. Isorhamnetin-based flavonoids presented higher antiproliferative activities than quercetin-based ones, while similar antioxidant properties were observed. The enzymatic acylation of isorhamnetin-3-O-glucoside with ethyl laurate and ethyl butyrate enhanced its capacity to inhibit xanthine oxidase and its antiproliferative activity but decreased its radical scavenging activity.     

Evaluation of antioxidant,antibacterial and anti-tyrosinase activities of four Macaranga species

The methanolic fresh leaf extracts of Macaranga gigantea, Macaranga pruinosa, Macaranga tanarius and Macaranga triloba were screened for their antioxidant properties (AOP), tyrosinase inhibition and antibacterial activities. Total phenolic content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, ferric-ion reducing power (FRAP), ferrous-ion chelating (FIC) and lipid peroxidation inhibition (LPI) activities were used to evaluate the AOP. Modified 3,4-dihydroxy-L-phenylalanine (L-DOPA) method was used to determine tyrosinase inhibition activity, whereas antibacterial activity was determined using the disc-diffusion technique. TPC screening of the same species from different collection sites showed no significant difference between sites. M.triloba showed the highest ascorbic acid equivalent antioxidant activity (AEAC), FRAP and LPI values. M. tanarius, which showed the lowest TPC, AEAC, FRAP and LPI activities, exhibited the best FIC activity. M. pruinosa showed the best tyrosinase inhibition activity, whereas M. triloba showed the best antibacterial activity against Gram-positive bacteria species, with minimal inhibition dosage (MID) values as low as 10 μg/disc.     

In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India

In this study, the antioxidant activity of methanol extracts of five plants from the genus Phyllanthus was evaluated by various antioxidant assays, including total antioxidant, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, nitric oxide scavenging, reducing power and metal ion chelating activities. The various antioxidant activities were compared to standard antioxidants such as butylated hydroxytoluene and ascorbic acid. All the extracts showed strong antioxidant activity in all the tested methods. Among the five plants Phyllanthus debilis has been found to possess the highest activity in all tested models, the activity decreased in the order Phyllanthus debilis>Phyllanthus urinaria>Phyllanthus virgatus>Phyllanthus maderaspatensis>Phyllanthus amarus. In addition to the antioxidant activity of these plants, the total phenolic compounds, flavonoids and flavonols were measured in the extracts. A correlation between the antioxidant activity and total phenolic content was observed.     

Phytochemical composition,anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece

The essential oils of four Teucrium species were studied and 150 components, in all, were identified. All oils were rich in sesquiterpenes (50.1–55.8%). Spathulenol and δ-cadinene were the main compounds of Teucrium brevifolium oil; caryophyllene and 4-vinyl guaiacol predominated in Teucrium flavum. Carvacrol and caryophyllene oxide predominated in Teucrium montbretii ssp. heliotropiifolium, while carvacrol and caryophyllene were the most abundant components in Teucrium polium ssp. capitatum. The oil which most effectively inhibited LPS-induced NO production in macrophage cell line RAW 264.7 was that from T. brevifolium (IC₅₀ = 7.1 μg/ml), followed by T. montbretii ssp. heliotropiifolium and T. polium ssp. capitatum (IC₅₀ = 16.5 and 29.4 μg/ml, respectively). The in vitro cytotoxic assay on three human cancer cell lines showed that the most antiproliferative oils were those from T. polium ssp. capitatum and T. montbretii ssp. heliotropiifolium on CACO-2 cell lines (IC₅₀ = 52.7 and 92.2 μg/ml, respectively). The T. brevifolium oil showed a selective cytotoxicity on COR-L23 while significant activity was exerted by T. polium oil on C32.     

Polysaccharides from Turbinaria conoides: Structural features and antioxidant capacity

Since reactive oxygen species (ROS) are involved in initiating and promoting several diseases such as cancer and cardiovascular events, this study was designed to evaluate the antioxidant activity of sulphated polysaccharides purified from Turbinaria conoides. A fucoidan (AF3), one alginic acid (B) and a neutral glucan (C1F2) were isolated. The glucan is made up of β-(1 → 3)-linked glucopyranosyl residues and has a molecular mass of 5 kDa. The alginic acid contains equimolar proportions of guluronic and mannuronic acid residues. The fucoidan is highly branched and has an average molecular mass of 50 kDa. The in vitro antioxidant capacity of the fractions was studied by ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. The fucoidan (AF3) showed the highest potential followed by the alginate (B) and laminaran (C1F2). Taken together, these findings demonstrate that these polysaccharides could be used as natural antioxidants by the food industry.