Purification,antitumor activity in vitro of steroidal glycoalkaloids from black nightshade (Solanum nigrum L.)

Six steroidal glycoalkaloids (1–6) were isolated and purified from Solanum nigrum L. (SNL) by acid extraction and alkaline precipitation, various chromatographic techniques, and their structures were elucidated by spectroscopic data. Antitumor activity, structure–activity and its molecular mechanism were investigated by methyl thiazolyl tetrazolium (MTT) method, flow cytometry, colorimetric assay and an immunocytochemical method. Experimental results showed that compounds 1 (solasonine), 2 (β1-solasonine), 3 (solamargine) and 6 (solanigroside P) have cytotoxicity to MGC-803 cells. Compounds with three sugar units and a-l-rhamnopyranose at C-2 or a hydroxyl group on the steroidal backbone may be potential candidates for the treatment of gastric cancer. The mechanism of action may be related to the decrease of mutation p53, the increase of the ratio of Bax to Bcl-2 and the activation of caspase-3 to induce apoptosis.     

Structural characterisation of polysaccharides from Tricholoma matsutake and their antioxidant and antitumour activities

In this study, polysaccharides from Tricholoma matsutake (TM-P) were purified using a DEAE-Sepharose fast flow column and three polysaccharide fractions (TM-P1, TM-P2 and TM-P3) were obtained. The chemical composition and structural characteristics of TM-P2 were quite different from those of TM-P1 and TM-P3. TM-P2 consisted of glucose, galactose and mannose with a molar ratio of 5.9:1.1:1.0. The glycosidic linkages were mainly composed of 1,6- and 1-linked glucose. Furthermore, TM-P2 showed the strongest in vitro antioxidant and antitumour activities. The oxygen radical absorbance capacity (ORAC) of TM-P2 was 2100.44 μmol Trolox/g. The antiproliferative activities of TM-P2 (4.0 mg/ml) on the growth of HepG2 and A549 cells were 67.98% and 59.04%, respectively.     

Response surface methodology applied to supercritical carbon dioxide extraction of Piper nigrum L. essential oil

This study was conducted to optimize the supercritical carbon dioxide (SC-CO₂) extraction of Piper nigrum L. essential oil using response surface methodology (RSM). In order to obtain the maximum yield of the essential oil, experiments were carried out using a three-factor central composite design (CCD) under following conditions: pressure of 15–30 MPa, temperature of 40–50 °C and dynamic extraction time of 40–80 min. A second-order polynomial regression model expressing the total extraction yield as a function of main SC-CO₂ variables was significantly (p < 0.05) fitted, with high coefficient of determination value (R² > 0.985). The results showed that the best extraction yield (2.16%) was obtained at 30 MPa, 50 °C and 80 min. Pressure showed the most significant (p < 0.05) effect on the yield variation. The chemical composition of the essential oil was determined using GC-flame ionization detection (FID) and gas chromatography–mass spectrometry (GC–MS) analysis. The main constituents (concentration > 3.0%, calculated as % peak area) in the P. nigrum L. essential oil obtained through SC-CO₂ extraction were determined to be β-caryophyllene (24.34%), limonene (15.84%), sabinene (15.04%), 3-carene (9.44%), β-pinene (9.27%) and copaene (4.52%).     

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.     

Spirostane and cholestane glycosides from the bulbs of Allium nigrum L

A phytochemical investigation of the fresh bulbs of Allium nigrum L. led to the isolation of new spirostane-type glycosides as two inseparable isomer mixtures, nigrosides A1/A2 (1a/1b) and nigrosides B1/B2 (2a/2b), two new cholestane-type glycosides, nigrosides C and D (3 and 4), together with the known compounds, 25(R,S)-5α-spirostan-2α,3β,6β-trio1-3-O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (5a/5b) and 25(R,S)-5α-spirostan-2α,3β,6β-trio1 3-O-β-d-glucopyranosyl-(1 → 2)-O-[4-O-(3S)-3-hydroxy-3-methylglutaryl-β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (6a/6b), isolated from this plant for the first time. All structures were elucidated mainly by spectroscopic analysis (1D and 2D NMR experiments, FABMS, HRESIMS) and by comparison with literature data. Cytotoxicity of the isolated compounds was assessed against human colon carcinoma (HT-29 and HCT 116) cell lines. Compounds 5a/5b and 6a/6b were found to be the most active with IC₅₀ values 1.09 and 2.82 μM against HT-29 and 1.59 and 3.45 μM against HCT 116, respectively.     

Antifungal,antiaflatoxin and antioxidant potential of chemically characterized Boswellia carterii Birdw essential oil and its in vivo practical applicability in preservation of Piper nigrum L. fruits

The study explores the chemical profile, antimicrobial and antioxidant activity of Boswellia carterii essential oil (EO). The EO significantly inhibited growth and aflatoxin production by the food borne toxigenic strain of Aspergillus flavus at 1.75 μl/ml and 1.25 μl/ml respectively. It exhibited broad fungitoxic spectrum against 12 food borne moulds and also showed strong antioxidant activity, IC₅₀ value and % inhibition of linoleic acid peroxidation being 0.64 μl/ml and 51.68% respectively. The antifungal action of EO was observed in terms of reduction in ergosterol content of plasma membrane of A. flavus. As fumigant in food system in storage containers, the EO provided 65.38% protection against fungal deterioration of Piper nigrum. GC–MS results revealed 31 components of EO. The chemically characterized B. carterii EO may thus be recommended as plant based preservative in view of its antifungal, antiaflatoxigenic, antioxidant activity and efficacy in food system.     

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.     

Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis

Exopolysaccharide (EPS) was isolated and purified from Lactococcus lactis subsp. Lactis culture broth. Selenium chloride oxide (SeCl₂O) was added to the EPS to synthesize selenium-exopolysaccharide (Se-EPS). The in vitro and in vivo antioxidant and in vivo immunomodulatory activity of EPS and Se-EPS were compared. EPS and Se-EPS scavenged superoxide anions and hydroxyl radicals. They also increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, while decreasing malondialdehyde (MDA) levels in serum and in the livers of mice. Se-EPS showed stronger in vitro and in vivo antioxidant activity than were shown by EPS. The in vivo immunoenhancement activity of EPS and Se-EPS induced by cyclophosphamide (CY) treatment in immunosuppressed mice was researched. EPS and Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and haemolytic complement activity (HC₅₀). Se-EPS showed stronger immunomodulatory activity than did EPS.     

Immunomodulatory and anticancer activities of phenolics from emblica fruit (Phyllanthus emblica L.)

There is a paucity of studies on the immunomodulatory properties of fruit extracts of emblica with the emphasis on lymphocytes. Therefore, the aim of the study was to evaluate the immunomodulatory properties and anticancer potential of six phenolic compounds from emblica fruit by in vitro proliferation assay. Effects of these compounds on splenocyte proliferation and the cytotoxicity to both human breast cancer cell (MCF-7) and human embryonic lung fibroblast cell (HELF) were determined by the MTT method. Significantly stimulatory effects (P < 0.05) were found for geraniin and isocorilagin. The concentration of geraniin, quercetin 3-β-d-glucopyranoside, kaempferol 3-β-d-glucopyranoside, isocorilagin, quercetin, kaempferol and rutin to obtain 50% of stimulatory effect was 56, 123, 242, 42, 73, 93 and 92 μg/ml, respectively. The assay of anticancer activities suggested that geraniin and isocorilagin exhibited higher cytotoxicities than other compounds against MCF-7 with IC₅₀ of 13.2 and 80.9 μg/ml, respectively. Isocorilagin exhibited a strong cytotoxicity to HELF cell with IC₅₀ of 51.4 μg/ml. Geraniin, quercetin, kaempferol and their glycosides had weak cytotoxicity against HELF cells. Paclitaxel showed a strong cytotoxicity to MCF-7 and HELF with IC₅₀ of 6.8 and 14.5 μg/ml, respectively. These findings are in line with the reported potent antioxidant activity. These results suggested that the antitumour activity of these compounds might be achieved by immunomodulatory properties which could partially be attributed to their antioxidant activity.     

Purification and characterisation of a hypoglycemic peptide from Momordica Charantia L. Var. abbreviata Ser.

A water-soluble peptide MC2-1-5 from Momordica charantia L. Var. Abbreviata Ser., with hypoglycemic effect, was purified by ultrafiltration, gel filtration chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The infrared (IR) spectra showed characteristic absorption peaks and the molecular mass of MC2-1-5 was found to be 3405.5174 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The sequence of its first 10 N-terminal amino acids was GHPYYSIKKS as determined by a protein sequencer. MC2-1-5 reduced the blood glucose level in alloxan-induced diabetic mice by 61.70% and 69.18% at 2 and 4 h, respectively, after oral administration at a dose of 2 mg/kg. The oral glucose tolerance test (OGTT) showed MC2-1-5 produced a reduction of 25.50%, 39.62% and 41.74% in blood glucose level after 1, 2 and 3 h, respectively, of oral administration compared with a diabetic control.     

Purification and characterisation of polyphenol oxidase from leaves of Cleome gynandra L.

A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6 kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60 °C, and stable from pH 3.0 to 9.0 and below 60 °C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg²⁺ and Pb²⁺, and completely inhibited by 1.0 mM of ascorbic acid, l-cysteine, β-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10 mM of dithioerythritol, sodium metabisulphite and sodium sulphite.     

Structure of a galactoarabinoglucuronoxylan from tamarillo (Solanum betaceum), a tropical exotic fruit,and its biological activity

Tamarillo (Solanum betaceum) is a tropical exotic fruit whose polysaccharides were extracted from the ripe pulp. After various purification steps, homogeneous fractions (designated PTW, STK-1000R and PF) were analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analysis. The results showed that the fraction PTW consisted of a linear arabinan with (1 → 5)-linked α-l-arabinofuranosyl units. Fractions designated as STK-1000R and PF contained galactoarabinoglucuronoxylans, with (1 → 4)-linked β-d-Xylp residues in the backbone, carrying branches exclusively at O-2. The polysaccharide in STK-1000R is less branched than that in the PF fraction (∼20.0% and 36.5%, respectively), with side-chains formed by (1 → 5)-linked α-l-Araf residues and (1 → 4)-linked α-d-GlcpA residues and with non-reducing end units formed by α-l-Araf, β-Arap, β-d-Galp, α-d-GlcpA and 4-O-Me-α-d-GlcpA. Intraperitoneal administration of the STK-1000R fraction in mice significantly reduced the number of abdominal constrictions induced by 0.6% acetic acid and the inflammatory phase of nociception induced by 2.5% formalin, indicating that that fraction has an antinociceptive effect on inflammatory pain models.     

Characterisation of leucyl aminopeptidase from Solanum tuberosum tuber

Potato juice (a waste product from the starch industry) is a potential source of novel enzymes for food applications. For use in the production and improvement of food protein hydrolysates, commercially available exopeptidases, predominantly aminopeptidases, are recommended. The present study was performed to explore possible biotechnological interest of leucyl aminopeptidase (LAP) activity in the potato tuber. The LAP from potato tuber was purified and characterised. Specific LAP activity was increased 200-fold by purification of the crude extract. The purified enzyme had a pH optimum of 9.0 and temperature optimum of 45 °C. LAP hydrolysed leucine-, alanine- and lysine-p-nitroanilide to a similar degree. The most efficient inhibitor was 1,10-phenanthroline. Almost all divalent cations tested inhibited the enzyme activity, while Co²⁺ stimulated LAP activity by over 100%. The purified LAP had a molecular weight of 90 kDa with an isoelectric point of 5.45. Sodium dodecylsulfate–polyacrylamide gel electrophoresis revealed one band of 48 kDa.     

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.     

In vivo immunomodulatory activity of polysaccharides derived from Chlorella pyrenoidosa

The effects of polysaccharides from Chlorella pyrenoidosa on immunomodulatory activity were investigated in this paper. Phagocytosis of peritoneal macrophages was detected through ingesting chicken red blood cells (CRBC) test to evaluate nonspecific immune response. The cellular immune response was measured using the delayed-type hypersensitivity response to sheep red blood cells (SRBC) in terms of antibody titration level. Oral administration of C. pyrenoidosa polysaccharides, 1 g and 2 g kg⁻¹ body weight significantly enhanced phagocytic rate (23.00 and 26.25%, respectively) and phagocytic index (0.30 and 0.36, respectively) compared with saline-treated control (P<0.05). The value of ear thickness rate was greatly elevated from 10.09% in saline-treated control to 16.22 and 19.48%, respectively in group treated with 1 g and 2 g kg⁻¹ body weight polysaccharides (P<0.05). There was a significant correlation between the dosage of C. pyrenoidosa polysaccharides and antibody titre (r=0.99, P=0.019) and the antibody titre was significantly increased by 11.80, 21.90 as well as 39.80 in 2 g kg⁻¹ bw compared to 10.30 in the control. Dietary supplementation with C. pyrenoidosa polysaccharides did not affect the growth of the mice during the experiment. These results indicated that immunomodulatory activities in mice were enhanced by administration of polysaccharides derived from C. pyrenoidosa.     

Chemical characterisation of anthocyanins in tamarillo (Solanum betaceum Cav.) and Andes berry (Rubus glaucus Benth.) fruits

The anthocyanin composition of tamarillo (Solanum betaceum Cav., red variety) and Andes berry (Rubus glaucus Benth.) was determined by HPLC–PDA and HPLC–ESIMS. From the anthocyanin-rich extracts (AREs), pure compounds (1–7) were obtained by MLCCC (multilayer countercurrent chromatography) and further preparative HPLC, and their unequivocal structures were obtained by 1D and 2D NMR analyses. The new anthocyanin delphinidin 3-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside-3′-O-β-d-glucopyranoside, as well as the known cyanidin-3-O-rutinoside, pelargonidin-3-O-rutinoside, and delphinidin-3-O-rutinoside were identified as constituents of tamarillo fruit. Although the anthocyanin composition of Andes berry had been reported before in the literature, the unequivocal structure elucidation of the major compound, cyanidin-3-O-α-l-rhamnopyranosyl-(1 → 6)-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside, was achieved for the first time.     

Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds

The seeds of Solanum dubium were blended and extracted using different types of buffers. The most reliable, quick, and efficient buffer was found to be 5% NaCl in acetate buffer (pH 5.0) which was used throughout the study. The extract was filtered and fractionated twice with ammonium sulphate. The partially purified enzyme was characterised by SDS–PAGE which showed a band of molecular weight of 66 kDa with the presence of other bands of low density. When compared with other plant enzymes, S. dubium enzyme was found to have higher clotting and proteolytic activities. The activity of the enzyme was steadily increased with enzyme and substrate concentration. The enzyme was found to be very stable against a wide range of pH values as well as a wide range of temperature (20–90 °C). The results of substrate specificity of the enzyme showed that the partially purified enzyme preferred both hydrophilic and hydrophobic amino acid residues at P1 position. The catalytic efficiency of the purified enzyme was enhanced by an aliphatic amino acids (Leu) compared to aromatic residue (Phe) at P1 position at the same site.