Characterisation and free radical scavenging activities of novel red pigment from Osmanthus fragrans’ seeds

A novel red pigment (RP) was isolated from Osmanthus fragrans  ’ seeds. The optimised experimental parameters of extraction obtained with a four-factor at three-level orthogonal array experimental design _L9(3⁴)$L_9(3^4)$ were ethanol concentration, temperature, pH and extraction time as 90%, 78 °C, 2.5 and 40 min, respectively. A yield of 34.6 ± 2.2 g/100 g was obtained under optimised conditions. The red pigment directly from O. fragrans’ seeds can be dissolved in alkaline, acidic waters solutions and hydrophilic organic solvents in common use. The colour of a water solution of RP changed with pH. RP was stable to heat in the temperature range of 25–100 °C. Physical and chemical properties of RP revealed that the red pigment was also stable in the presence of Na₂SO₃, NaCl, amino acid, organic acid, sugar, starch or metal-ions (such as Ca²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Al³⁺, Mg²⁺ and Na⁺), but was bleached by strong oxidants (KMnO₄, K₂Cr₂O₇ and NaOCl). Subsequently, free radical scavenging activities of RP were assessed using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals using a new resonance scattering spectral method. RP showed an excellent DPPH radical scavenging activity and was superior to butylated hydroxytoluene (BHT), and exhibited quite a strong concentration-dependent inhibition of hydroxyl radical at low concentrations compared with ascorbic acid and quercetin. When the concentration of RP was 0.03 μg/ml, the scavenging percentage of hydroxyl radical reached 92.3%. Salidroside was isolated as an active principle.     

桂花种子皮黑色素提取及影响其稳定性因素分析

采用碱性溶液提取、酸水解、多种有机溶剂萃取沉淀等工艺手段分离纯化了桂花种子皮色素,呈天然黑色,其得率为0.34%(以湿原料为准)。随后,对该色素的理化性质等进行了较系统的研究。结果表明,桂花种子皮黑色素耐光、耐热;对还原剂较稳定;对强氧化荆不够稳定;碱性条件下较稳定;Cu2 、Ca2 、Fe3 、Zn2 等金属离子对该色素有护色、增色作用;Mg2 、Al3 、Na 虽有减色作用,但不明显, 是一种性能优良的天然黑色素。     

Antioxidant activity and melanogenesis inhibitory effect of the acetonic extract of Osmanthus fragrans: A potential natural and functional food flavor additive

Osmanthus fragrans, a common flavor additive for tea and other beverages, has potential applications in biomedical science. In this study, we investigated O. fragrans acetonic extract (OFE) for its total phenolic and flavonoid contents, radical-scavenging activity, and potential anti-tyrosinase ability. OFE possessed considerable amounts of phenolics (264.7 mg gallic acid equivalent/g of extract) and flavonoids (190.7 mg catechin equivalent/g of extract). The antioxidation activities, measured in terms of EC₅₀ values using DPPH and ABTS⁺ assays, were 304.9 mg ascorbic acid equivalent/g of extract and 516.3 mg trolox equivalent/g of extract, respectively. LC-MS results discovered the presence of luteolin in the extract, and a kinetic study revealed an uncompetitive inhibitory effect of OFE upon the oxidation of tyrosine (IC₅₀ = 2.314 mg/mL) and l-DOPA (IC₅₀ = 44.20 mg/mL). In addition, we tested OFE in vitro (B16F10 cells) for its anti-tyrosinase activity and anti-melanin formation and found that OFE was able to reduce the tyrosinase activity and melanin formation of B16F10 cells in a dose-dependent manner. Our findings support that O. fragrans is a potential natural, functional antioxidant food favor additive. Additionally, because OFE inhibits melanin formation, it appears to have potential use as an anti-browning food additive, in skin-whitening cosmetics, or as a new drug for treating melanoma.     

Isolation and characterization of pigment from Cinnamomum burmannii' peel

Cinnamomum burmannii is cultivated for use as a spice, and as an ornamental tree. With the aim to develop a natural colorant for use in cosmetics and food additives from a new source, in the present study, pigment derived from C. burmannii' peel (CBP) was isolated by alkaline extraction, acid hydrolysis, repeated precipitation and purification by Sephadex G-75 with a total yield of 0.06 g/100 g (wet weight basis). The color values of the pigment was E_1 cm^1% 278 nm = 65.21. Physical and chemical properties revealed that CBP presses similar properties as most natural pigment. It was scarcely soluble in both water and all common organic solvents, and was soluble only in alkaline aqueous and DMSO. It was stable under ultraviolet light or room-light, stable in the range of 25-100 °C, relatively stable in alkaline aqueous and reducer, but was bleached by strong oxidants (KMnO₄, H₂O₂ and NaOCl). Sodium benzoic acid, tartaric acid, table salt and cane sugar affected it slightly. Spectroscopic analysis of CBP in relation with its structure was also discussed. This is the first report on the characterization of pigment obtained from C. burmannii 'peel.     

Variations in the components of Osmanthus fragrans Lour. essential oil at different stages of flowering

A headspace solid-phase microextraction (HS-SPME) method was used to extract the essential oil of Osmanthus fragrans Lour., which was then analysed by gas chromatography–mass spectrometry (GC–MS) at four different stages of flowering. The primary chemical components of the essential oil extracted from O. fragrans were linalool and its oxide, α-ionone, β-ionone, nerol, γ-decalactone, 9,12,15-octadecatrienoic acid, and hexadecanoic acid, most of which were at their highest concentrations in extracts obtained from flowers at the initial flowering stage. HS-SPME using a PDMS/DVB fibre is a simple, rapid, and solvent-free method for the extraction of volatile compounds emitted from living O. fragrans. HS-SPME with GC–MS can be used to determine the aromatic maturity in O.fragrans, to decide the optimal harvest date. The initial flowering stage was the best time to harvest O. fragrans.     

Antioxidant capacity of the extracts from pulp of Osmanthus fragrans and its components

In this study, a significant inhibitory effect (EC₅₀ = 50.29 μg/ml) on the ABTS free radicals was detected in the crude ethanol extracts (CEE) of Osmanthus fragrans' pulp. Then, CEE was separated into methanol soluble fraction (MSF) and a methanol insoluble fraction (MISF), and their scavenging activities on the ABTS free radicals ascended in the order: MSF > CEE > MISF, respectively. Accordingly, the total phenolic (TP) contents of MSF, CEE and MISF were 40.53 ± 0.27, 35.37 ± 0.18 and 29.62 ± 0.23 mg/g (dry weight), respectively. Furthermore, salidroside (tyrosol 8-O-β-glucopyranoside), which showed powerful antioxidant activities in all test system was isolated from MSF. The antioxidant activity of salidroside (81.54 μg/ml) on DPPH free radicals was found to be nearly twice of that of MSF (144.78 μg/ml), and its total antioxidant activity is superior to that of the synthetic antioxidant butylated hydroxytoluene (BHT). In conclusion, our results suggested that O. fragrans' pulp, which was often considered as agricultural wastes, could be a promising source of natural antioxidants, and that MSF and salidroside may be good candidates for further development as natural antioxidants.     

Isolation, purification and characterization of a new gum from Acanthophyllum bracteatum roots

A new gum was isolated from the roots of Acanthophyllum bracteatum (ABG) by warm-water extraction. Purification was carried out by barium complexing to give a yield of 12.4% of pure air-dried or 5.8% of freeze-dried gum. The ABG contained 13.2% moisture, 84.3% carbohydrate, 0.9% protein and 1.5% ash. Its mineral content was comparable to commercial hydrocolloids. Monosaccharide analysis by HPLC showed the presence of galactose, glucose, arabinose, rhamnose and uronic acids in the ratio 16.0:7.2:3.0:1.0:3.1 respectively. The viscosity and pH value of 1% ABG solution at 25 °C were 51.5 mPa s and 6.85 respectively. ABG solutions (5-30 wt%) showed shear-thinning flow behavior at shear rates < 10 s⁻¹. The viscosity decreased as temperature increased, and was highest at the neutral state. ABG had low surface and emulsification properties but moderate foaming capacity and relatively high foaming stability, which suggests that ABG could potentially be used in food systems to improve foaming properties.     

Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds

A plant lectin isolated in its pure state from the Egyptian seeds of Pisum sativum (PSL) produced two bands in SDS–PAGE (5.53 and 19.3 kDa; i.e. α and β chain) but one peak by gel filtration chromatography on Sephadex G-100, corresponding to 50 kDa, i.e., a dimeric structure of two monomers, each consisting of one α and one β subunit. PSL is a glycoprotein bound with glucose (2 mol/mol of protein) and stabilized by 2 atoms of each of Ca²⁺ and Mn²⁺ per molecule of protein. It highly agglutinated human, rabbit and rat erythrocytes but weakly agglutinated chicken erythrocytes, while no agglutination occurred with sheep erythrocytes. Hemagglutination was markedly affected by acidic pH, but was heat stable below 60 °C for 30 min. Among the various tested sugars, PSL agglutination was most inhibited by mannose. PSL is rich in hydroxyl amino acids while totally lacking sulfur amino acids. PSL inhibited the growth of Aspergillus flavus, Trichoderma viride and Fusarium oxysporum.     

Physicochemical characterization and functional properties of galactomannans from mesquite seeds (Prosopis spp.)

In the present study, the physicochemical and functional characteristics of galactomannans (GM) from mesquite seeds were investigated and compared with the characteristics of a commercial GM. The mesquite gum was extracted with water at room temperature, and its physicochemical parameters, thermal constants and functional properties were determined. The results showed that mesquite GM had contents of 2.56% moisture, 4.54% protein and 0.06% ash; trace levels of fat; 95.40% total carbohydrate; +63.48 optical rotation; an intrinsic viscosity of 12.36 dL/g and a molecular weight of 1.2 × 10⁶ g/mol. The sugars detected were mannose (Man) and galactose (Gal), with a Man:Gal ratio of 1.50. The curves obtained by calorimetry indicated a transition temperature (Tg), melting temperature (Tm) and heat capacity (Cp) very similar to those of the guar GM. The evaluation of the functional properties of the mesquite GM revealed a solubility of 92.10%, an emulsion capacity of 95% and an emulsion stability of 92.24%. The polysaccharide extracted from the mesquite seeds is a GM with physicochemical and functional properties similar to those reported for other legume seed gums, allowing us to conclude that mesquite GM has the potential for use in the food industry.     

Composition and antimicrobial activity of essential oils of Cinnamosma fragrans

Essential oil samples of Cinnamosma fragrans from two regions in Madagascar, Tsaramandroso (38 samples) and Mariarano (30 samples), were analysed by GC/MS. Fifty-seven components were identified, accounting from 88.3% to 99.4% of the oils’ composition. The major components were linalool (72.5 ± 23.3%) in Tsaramandroso and 1,8-cineole (47.3 ± 10.2%) in Mariarano.     

Solvent-free microwave extraction of essential oil from Dryopteris fragrans and evaluation of antioxidant activity

Solvent-free microwave extraction (SFME) of the essential oil from Dryopteris fragrans and its antioxidant activity were investigated. A central composite design combined with response surface methodology was applied to study the influences of extraction time, irradiation power and humidity (proportion of water pretreatment). A maximal extraction yield of 0.33% was achieved under optimal conditions of extraction time 34 min, irradiation power 520 W and humidity 51%. Sixteen compounds, representing 89.65% of the oil, were identified, of which the major ones, (1R,4S,11R)-4,6,6,11-tetramethyltricyclo[5.4.0.0(4,8)]undecan-1-ol (30.49%), 1R,4S,7S,11R-2,2,4,8-tetramethyltricyclo[5.3.1.0(4,11)]undec-8-ene (22.91%) and, 1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethyl-1-naphthalenemethanol (15.11%), accounted for 68.51% of the oil. The antioxidant activity of the essential oil was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene/linoleic acid, and reducing power assay, the IC₅₀ values were 0.19, 0.09 and 0.18 mg/mL, respectively. All these results suggest that SFME represents an excellent alternative protocol for production of essential oils from plant materials.     

Isolation,characterization, and neuroprotective activities of sesquiterpenes from Petasites japonicus

Neuroprotective reagents to protect the nerve cells against oxidative stress and other damages are potentially effective for the medical treatment of Parkinson’s disease. Petasites japonicus, a wild vegetable, belongs to the family Compositae and its extract has shown the neuroprotective effects. A further phytochemical investigation of P. japonicus for neuroprotective substances led to the isolation of eight new (1–8) and two known (9 and 10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, ¹H–¹H COSY, and NOESY) spectroscopic data analyses, and the structure of 1 was confirmed by X-ray crystallography. The neuroprotective activities of these sesquiterpenes were evaluated against cobalt chloride (CoCl₂)-induced neuronal cell death in human dopaminergic SH-SY5Y cells. Five compounds showed a neuroprotective activity.     

Microwave-assisted ionic liquids pretreatment followed by hydro-distillation for the efficient extraction of essential oil from Dryopteris fragrans and evaluation of its antioxidant efficacy in sunflower oil storage

With strong dissolution abilities of ionic liquids to plant cell walls, an integrated microwave-assisted ionic liquids pretreatment followed by hydro-distillation method (MILP–MHD) was performed for the efficient extraction of essential oil from the rare Dryopteris fragrans. Under optimal conditions, this innovative MILP–MHD provided the remarkable superiority of much higher yield (0.91%) of D. fragrans essential oil (DEO) within much shorter extraction duration (14.2 min) than those of the reported solvent-free microwave extraction technique (0.33% and 94 min). Furthermore, the antioxidant efficacy of DEO in the stabilization of sunflower oil was determined and found to be comparable to the commonly-employed α-tocopherol (VE) but a little lower than the synthetic antioxidant butylated hydroxytoluene (BHT). Therefore, MILP–MHD is a potential and highly efficient technique for the extraction of valuable essential oils in flavor food industries and DEO can be explored as a potent natural antioxidant additive for edible oils or lipid foods storage.     

Purification and characterization of membrane-bound polyphenoloxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss]

Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40–80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS–PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38 kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30 °C and active towards diphenols as substrates. The K_m and V_max values were calculated to be 5.46 mM and 0.98 U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC₅₀ of 1.3 ± 0.002 mM followed by ascorbic acid (1.5 ± 0.06 mM), glutathione (1.5 ± 0.07 mM), EDTA (100 ± 0.02 mM) and citric acid (186 ± 0.16 mM).     

Isolation and characterization of galactomannan from Dimorphandra gardneriana Tul. seeds as a potential guar gum substitute

A galactomannan was obtained from mature seeds of Dimorphandra gardneriana Tul., the plant from which rutin is extracted. The galactomannan extraction was based on manual separation of the endosperm, water dissolution, centrifugation and precipitation with ethanol. The galactomannan yield obtained (31%) was similar to values reported for other Brazilian seeds and to that of guar gum. The polysaccharide from D. gardneriana seeds (GalDG) was characterized by gas–liquid chromatography (GLC), gel permeation chromatography (GPC), rheology and also by ¹³C and ¹H nuclear magnetic resonance (NMR). The monosaccharide composition in weight % was mannose 64.2, galactose 34.7 and glucose 1.1. Small amounts of protein and uronic acid were found, values being 1.75 and 2.8% (w/w), respectively. The mannose/galactose ratio of GalDG (1.84) is similar to values reported for galactomannans extracted from other Brazilian seeds, and is the M/G value closest to that of guar gum (1.6–1.8). The intrinsic viscosity of galactomannan from D. gardneriana (8.7 dL/g), in water at 25 °C, is lower than the [η] value of guar gum, but the absolute viscosity of the GalDG in aqueous solution at concentrations of 0.1 and 1% (w/v) is higher. The aqueous solution at 1% (w/v) behaves as a pseudoplastic fluid, but a Newtonian behavior was noted for the solution at 0.1%. The high average molar masses, M_w of 3.9 × 10⁷ g/mol and M_n of 1.9 × 10⁷ g/mol, determined by GPC are probably due to molecular aggregation. ¹³C and ¹H NMR spectra (DEPT 135 and HSQC) of GalDG solutions in D₂O were recorded. The patterns of mannose substitution in GalDG and guar gum are similar.     

Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.)

Lipoxygenase (LOX) from pea seeds (Pisum sativum var. Telephone L.) was extracted and studied of biochemical properties. The molecular mass of purified lipoxygenase was 93 kDa. The effects of substrate specificity, pH, and sensibility to various inhibitors: caffeic acid, ferulic acid, benzoic acid, catechin, quercetin and kaempferol of LOX were investigated. Lipoxygenase showed the highest activity toward linoleic acid and the lowest toward oleic acid as substrates. Kinetic studies indicated that V_max of the LOX activity was 151.5 U/min and corresponding K_m value of 0.44 × 10⁻³ M. Optimum pH of lipoxygenase was reported at 5.5. Caffeic acid was the most effective inhibitor and kaempferol was the least effective.     

Spectrophotometric determination of total serotonin derivatives in the safflower seeds with Ehrlich’s reagent and the underlying color reaction mechanism

A new spectrophotometric method for the determination of total serotonin derivatives in the safflower (Carthamus tinctorius L.) seeds is described. The determination is based upon a color reaction between serotonin derivatives and p-dimethylaminobenzaldehyde (Ehrlich’s reagent), which follows the electrophilic substitution reaction mechanism at the indole ring. The main factors affecting correct measurement of total serotonin derivatives concentration were studied. The maximum absorption wavelength of the complex was determined at 625 nm. Lambert-Beer’s law is obeyed in the concentration range of 0.025–0.5 mmol/l, with a correlation coefficient (R²) of 0.9996, a recovery of 99.7%, and a relative standard derivation (RSD) of 1.5%, respectively. The proposed method presented satisfactory results in the determination of total serotonin derivatives in the extract from a strain of safflower seeds, and thus is recommended as a routine method for total serotonin derivatives quantitation.     

Chemical constituents with antioxidant activities from litchi (Litchi chinensis Sonn.) seeds

Litchi (Litchi chinensis Sonn.) is widely accepted as a delicious fruit in China and its seeds have been commonly used in traditional Chinese medicine to relieve neuralgic pain. In the present study, chemical investigation of the 95% ethanol extract of Litchi chinensis seeds led to the isolation of four new compounds, 2α,3α-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4β-8-catechin) (5), 2β,3β-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4α-8-epicatechin) (7), litchiol A (9) and litchiol B (12), together with 11 known ones, 2,5-dihydroxy-hexanoic acid (1), soscopoletin (2), coumaric acid (3), protocatechuic acid (4), 2α,3α-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4β−8)-epicatechin (6), pterodontriol d-6-O-β-d-glucopyranoside (8), Narirutin (10), naringin (11), dihydrocharcone-4′-O-β-d-glucopyranoside (13), pinocembrin-7-rutinoside (14), pinocembrin-7-neohesperidoside (15). Their structures were mainly elucidated on the basis of NMR, MS, IR, CD and UV spectral evidences. Antioxidant activities of 14 compounds were determined by DPPH radical-scavenging assay and Trolox equivalent antioxidant capacity assay, and the results showed that four compounds, protocatechuic acid (4), 2α,3α-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4β-8-catechin (5), 2α,3α-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4β−8)-epicatechin (6), 2β,3β-epoxy-5,7,3′,4′-tetrahydroxyflavan-(4α-8)-epicatechin (7), exhibited moderate antioxidant activities.     

Isolation, characterization of melanin derived from Ophiocordyceps sinensis, an entomogenous fungus endemic to the Tibetan Plateau

Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds. In this present study, a black pigment was isolated from the fermentation broth of Ophiocordyceps sinensis, an entomogenous fungus which is endemic to the Tibetan Plateau by alkaline extraction, acid hydrolysis, and repeated precipitation. It was designed as melanin since the physical and chemical properties including its ultraviolet (UV) and infrared (IR) spectra of the black pigment conformed to the characteristic of melanin and similar to the commercial synthetic melanin. The antioxidant activity of melanin derived from O. sinensis was evaluated. They showed much stronger scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and the chelating ability on ferrous ions than that of the water extract from the mycelia of O. sinensis, with IC₅₀ value 18.51 ± 0.85 μg/ml and 2.58 ± 0.26 μg/ml, separately. This is the first report of melanin from O. sinensis and will be helpful for the study on the physiology and the artificial cultivation of this fungus, an endangered species.