Antioxidant activity of different species and varieties of turmeric (Curcuma spp): Isolation of active compounds.

There are >80 species of turmeric (Curcuma spp.) and some species have multiple varieties, for example, Curcuma longa (C. longa) has 70 varieties. They could be different in their chemical properties and biological activities. Therefore, we compared antioxidant activity, total phenolic and flavonoid content of different species and varieties of turmeric namely C. longa [variety: Ryudai gold (RD) and Okinawa ukon], C. xanthorrhiza, C. aromatica, C. amada, and C. zedoaria. The antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, oxygen radical absorbance capacity (ORAC), reducing power and 2-deoxyribose (2-DR) oxidation assay. Our results suggested that RD contained significantly higher concentrations of total phenolic (157.4 mg gallic acid equivalent/g extract) and flavonoids (1089.5 mg rutin equivalent/g extract). RD also showed significantly higher DPPH radical-scavenging activity (IC50: 26.4 µg/mL), ORAC (14,090 µmol Trolox equivalent/g extract), reducing power absorbance (0.33) and hydroxyl radical scavenging activity (IC50: 7.4 µg/mL). Therefore, RD was chosen for the isolation of antioxidant compounds using silica gel column, Toyopearl HW-40F column, and high-performance liquid chromatography. Structural identification of the compounds was conducted using 1H NMR, 13C NMR, and liquid chromatography-tandem mass spectrometry. The purified antioxidant compounds were bisabolone-9-one (1), 4-methyllene-5-hydroxybisabola-2,10-diene-9-one (2), turmeronol B (3), 5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-1-hepten-3-one (4), 3-hydroxy-1,7-bis(4-hydroxyphenyl)-6-hepten-1,5-dione (5), cyclobisdemethoxycurcumin (6), bisdemethoxycurcumin (7), demethoxycurcumin (8) and curcumin (9). The IC50 for DPPH radical-scavenging activity were 474, 621, 234, 29, 39, 257, 198, 47 and 18 µM and hydroxyl radical-scavenging activity were 25.1, 24.4, 20.2, 2.1, 5.1, 17.2, 7.2, 3.3 and 1.5 µM for compound 1, 2, 3, 4, 5, 6, 7, 8 and 9, respectively. Our findings suggested that the RD variety of C. longa, developed by the University of the Ryukyus, Okinawa, Japan, is a promising source of natural antioxidants.

Label-free proteomic analysis to characterize ginger from China and Ghana.

Ginger is a popular spice used in food and beverages. In this study, we sought to characterize and differentiate ginger samples of Ghana and China origin using label-free proteomic and untargeted metabolomic analyses. As result, a total of 180 proteins significantly changed between the ginger samples from both studied countries. Among them, 17 proteins were specifically identified in the Chinese ginger, while 23 proteins were only identified in the Ghanaian ginger. Function and bioinformatics analyses indicated that changes in carbon metabolism, secondary metabolites biosyntheses, citrate acid cycle, and amino acids biosyntheses-related pathways contributed to the differences. These results were confirmed through the identification of 14 significantly changed metabolites including diarylheptanoids and gingerols. Importantly, change tendencies of these metabolites corresponded to changes in abundance of the protein enzymes involved in their syntheses. These results suggest that changes in metabolism-related protein enzymes are responsible for the intraspecies difference of the ginger samples.

Determination of the Marker Diarylheptanoid Phytoestrogens in Curcuma comosa Rhizomes and Selected Herbal Medicinal Products by HPLC-DAD.

A method for quantification of diarylheptanoids in Curcuma comosa rhizomes and selected pharmaceutical preparations was established by using HPLC-diode array detector (DAD). The chromatographic separation of three diarylheptanoids [(3S)-1-(3,4-dihydroxy-phenyl)-7-phenyl-(6E)-6-hepten-3-ol (1), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (2), and (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (3)] was performed on a Luna C18 analytical column using gradient elution with 0.5% acetic acid in water and acetonitrile with a flow rate of 1 mL/min and a column temperature of 35°C. The calibration curves for the analytes showed good linearity (R2>0.999), high precision (relative standard deviation (RSD) <2%) and acceptable recovery (98.35-103.90%, RSD <2%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.06-0.22 and 0.18-0.69 µg/mL, respectively. The results of all validated parameters were within the limits according to the International Conference on Harmonization (ICH) Guidelines. The established method was successfully applied for qualitative and quantitative determination of the three constituents in different samples of C. comosa and some commercial products in capsules. The simplicity, rapidity, and reliability of the method could be useful for the fingerprint analysis and standardization of diarylheptanoids, which are responsible for the estrogenic activity in raw materials and herbal medicinal products of C. comosa.

[Study on diarylheptanoids from green peel of Juglans sigillata].

The chemical constituents from green peel of Juglans sigillata were isolated by column chomatographies over silica gel, Sephadex LH-20, and MCI. Four diarylheptanoids were isolated and their structures were characterized as dihydropterocarine(1), 3',4″-epoxy-1-(4'-hydroxy-phenyl)-7-(3″-methoxyl-phenyl)-heptan-3α-ol(2), pterocarine(3), and 1-(4'-hydroxy-phenyl)-7-(3″-methoxy-4″-hydroxyphenyl)-heptan-3α-ol(4). Compound 1 is a new compound, named as dihydropterocarine. Compounds 2-4 were isolated from the plant of J. sigillata for the first time.

Simultaneous quantification of diarylheptanoids in Alnus nepalensis using a validated HPTLC method.

Platyphylloside, oregonin and hirsutanonol 5-O-ß-D-glucopyranoside are known bioactive metabolites in Alnus nepalensis. In this article, the aforementioned three markers were isolated and simultaneously quantified by the thin-layer chromatography densitometric method. A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin-layer chromatographic method was developed and validated for the determination of above markers in the leaves of A. nepalensis. The separation was performed on silica gel 60F254 high-performance thin-layer chromatography plates using chloroform:methanol:formic acid (75:25:2, v/v) as mobile phase. The quantitation of diarylheptanoids was carried out using the densitometric reflection/absorption mode at 610 nm after post-chromatographic derivatization with vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 333-3330 ng/spot with good correlation (r(2) = 0.999). The method was validated for peak purities, precision, robustness, limit of detection and quantitation, etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor and spectra correlation of markers in standard and sample tracks.

Characterisation of diarylheptanoid- and flavonoid-type phenolics in Corylus avellana L. leaves and bark by HPLC/DAD-ESI/MS.

INTRODUCTION: The leaves of Corylus avellana L. (common hazel, Betulaceae), a plant with a wide distribution in Europe, have been used in folk medicine for various diseases, but phytochemical exploration of C. avellana is still incomplete. To the best of our knowledge there is no previous report concerning diarylheptanoids in C. avellana, although these compounds show a frequent occurrence among Betulaceae plants. OBJECTIVE: To improve existing online chromatographic methods for the investigation of the phenolic compounds in C. avellana leaves and bark, focusing on diarylheptanoid-type molecules. METHODS: Dried and powdered leaves and bark of C. avellana were extracted with increasing polarity solvents (n-hexane, chloroform, ethyl acetate and methanol) in Soxhlet extractor apparatus. For the characterisation of the phenolic compounds in the ethyl acetate and methanolic extracts, UV spectral data, obtained by LC with a diode-array detector (DAD), accurate molecular mass and formula, acquired by LC and electrospray ionisation (ESI) with time-of-flight (TOF) MS and fragmentation pattern, given by LC-ESI/MS/MS analyses were used. Quantitation of the compounds was performed by LC-MS/MS. RESULTS: In the methanolic and ethyl acetate extracts of C. avellana bark four flavonoid glycosides and a caffeoyl hexoside derivative were detected and characterised, while in C. avellana leaves, seven diarylheptanoid-type molecules were tentatively identified in addition to six flavonoid components. As far as we know this is the first study where the presence of diarylheptanoids in C. avellana is reported. CONCLUSION: The improved HPLC/DAD-ESI/MS method was successfully utilised for the characterisation and quantitation of the phenolic compounds in C. avellana bark and leaves extracts.

One new and several minor diarylheptanoids from Amomum muricarpum.

A new natural diarylheptanoid, designated muricarpin, together with four diarylheptanoids were isolated from the rhizomes of Amomum muricarpum Elmer (Zingiberaceae) growing in Vietnam. Three known compounds, 1,7-bis(3,4-dihydroxyphenyl)heptan-3-yl acetate, 1-(4'-hydroxyphenyl)-7- (3″,4″-dihydroxyphenyl)heptan-3-yl acetate and 1-(3',4'-dihydroxyphenyl)-7-(4″-hydroxyphenyl)-heptan-3-one were isolated for the first time from the genus Amomum, meanwhile (5R)-5-hydroxy-1,7-bis(4-hydroxyphenyl)-heptan-3-one was found for the first time in plants. Their structures were determined using spectroscopic analyses.

Separation and identification of diarylheptanoids in supercritical fluid extract of Alpinia officinarum by UPLC-MS-MS.

In the present study, ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization (ESI(+)) tandem mass spectrometry (MS) was developed to identify and characterize the diarylheptanoids in the supercritical fluid extract (SFE) of Alpinia officinarum. The method established provides good reproducibility of UPLC and shows high precision with all the mass accuracy of less than 5 ppm. The ESI-MS-MS fragmentation behavior of every group and their appropriate characteristic pathways were proposed. On the basis of analyzing the fragmentation pathways, elemental composition provided by software Masslynx, mass data of the standard compounds and the information regarding polarity obtained from retention time data, in all, 23 diarylheptanods were characterized. All of them have been reported in Alpinia officinarum. They were classified into six distinct groups (homologous series). Compared to the references, the fragmentation pathways of the first and second group were detailed much more and complementary. Further more, the fragmentation pathways of the last four groups were firstly discussed. The fragmentation rules deduced and the data provided could aid in the characterization of other diarylheptanoids of these types and would be useful for the further research of diarylheptanoids in Alpinia officinarum or the other plants.

A new diarylheptanoid glycoside from the stem bark of Alnus hirsuta and protective effects of diarylheptanoid derivatives in human HepG2 cells.

To search for secondary metabolites of Alnus hirsuta (Betulaceae), various chromatographic separations of the ethyl acetate soluble fraction of the stem bark of A. hirsuta led to the isolation of a new diarylheptanoid glycoside, (3R)-1,7-bis-(4-dihydroxyphenyl)-3-heptanol 3-O-beta-D-glucopyranosyl(1-->3)-beta-D-xylopyranoside (13) and twelve diarylheptanoid derivatives, namely, oregonin (1), rubranoside A (2), hirsutanonol 5-O-beta-D-glucopyranoside (3), rubranoside B (4), rubranoside C (5), hirsutanonol (6), hirsutenone (7), (5S)-O-methylhirsutanonol (8), platyphylloside (9), platyphyllonol 5-O-beta-D-xylopyranoside (10), aceroside VII (11) and platyphyllenone (12). Isolates were assessed for their hepatoprotective effects against tert-butylhydroperoxide (t-BHP)-induced toxicity in HepG2 cells. Of these isolates, compounds 1-8 showed significant hepatoprotective effects on t-BHP-induced damage to HepG2 cells, with 8 exhibiting the greatest protective effect (50.7 + or - 3.7% at a concentration of 10 microM).

Capillary zone electrophoresis for separation and analysis of four diarylheptanoids and an alpha-tetralone derivative in the green walnut husks (Juglans regia L.).

A fast capillary zone electrophoresis (CZE) method for the simultaneous determination of four cyclic diarylheptanoids (rhoiptelol, RH; juglanin A, JA; juglanin B, JB; juglanin C, JC) and an alpha-tetralone derivative (sclerone, SC) in the extract of the green walnut husks (Juglans regia L.) was developed. The optimized buffer was composed of 25 mM sodium tetraborate at pH 10.3. The applied voltage was 20 kV and the capillary temperature was kept constant at 20 degrees C. The detection wavelength was set at 220 nm using a photodiode array detection. The effects of several CE parameters, including pH value, buffer concentration, applied voltage and separation temperature on the separation were investigated systematically. Regression equations showed good linear relationships (correlation coefficients: 0.9996-0.9999) between the peak area of each compound (RH, JA, JB, JC and SC) and its concentration accordingly. The relative standard deviations (R.S.D.) of the migration time and peak area were less than 0.57 and 3.44% (intra-day), and 0.97 and 3.71% (inter-day), respectively. The contents of the five active compounds in the green walnut husks (J. regia L.) from different origins were determined with satisfactory repeatability and recovery.

Simultaneous determination of three diarylheptanoids and an alpha-tetralone derivative in the green walnut husks (Juglans regia L.) by high-performance liquid chromatography with photodiode array detector.

By optimizing extraction, separation and analytical conditions, a reliable and accurate high-performance liquid chromatographic (HPLC) method coupled with photodiode array detector (DAD) at room temperature is developed for simultaneous determination of three diarylheptanoids (juglanin A, juglanin B, rhoiptelol) and an alpha-tetralone derivative (regiolone) in methanol extracts from the green walnut husks (Juglans regia L.) The sample pretreatment process involved the reflux extraction using methanol as the extract with a ratio of liquor to sample of 15 mL/g. The separation was achieved on a SinoChrom ODS-AP C(18) column with gradient elution using acetonitrile and 2% (v/v) acetic acid in water. The intra-day and inter-day precision (RSD%) for the analytes ranged from 1.08 to 1.51 and 0.60 to 1.13, respectively. The average recoveries obtained were from 88.4% to 96.2% for the analytes with RSDs below 3.13%. The correlation coefficients of the calibration curve exceeded 0.999. The detection limits were 0.51, 0.25, 0.32 and 0.35 ng at a signal-to-noise ratio of 3, respectively. Quantitative analyses of the samples from different grown sites and in obtained different months showed that the contents of the analytes varied significantly. The method was then successfully applied for the detection and isolation of a new diarylheptanoid derivative in the green walnut husks (J. regia L.). The structure of the new compound was elucidated by various spectroscopic methods including 2D NMR techniques (COSY, HMQC, HMBC), HR-ESI-MS and X-ray single-crystal diffraction analysis.

Determination of oregonin in Alnus plants and biological samples by capillary electrophoresis.

Oregonin, existing primarily in the Alnus plants, displayed anti-inflammatory and antioxidative activities. The capillary zone electrophoresis (CZE) method was developed in this study to quantitatively determine oregonin content in the Alnus plants for the first time. Various parameters, including buffer concentration, pH and applied voltage, were evaluated for their optimum analytical conditions. The optimized buffer was composed of 30 mM sodium tetraborate at pH 8.0. The separation voltage was set at 30 kV and the UV detection wavelength was set at 220 nm. Oregonin could be determined within 6 min under such optimized conditions. Relative standard deviation (R.S.D.) of the run-to-run repeatability and intermediate precision of the retention time of oregonin was within 1.36%. Run-to-run repeatability and intermediate precision of the peak area ratios of oregonin to internal standard, theophylline, were both within 1.55% R.S.D. The presented method was applied to analyze oregonin in leaves of Alnus formosana, seeds of various Alnus plants as well as biological samples. The stability of oregonin in biological system was indicated in this study. It demonstrates the potential of this developed method in natural product research.

NMR-based metabolic profiling of Anigozanthos floral nectar.

Nuclear magnetic resonance spectroscopic methods have been used to characterize the chemical composition of floral nectar of Anigozanthos species with a minimum of sample preparation and without derivatization. The nectar of this passerine-pollinated plant is largely dominated by glucose and fructose, while sucrose occurs only at a minor level. Tyrosine, several additional amino acids, and a variety of carboxylic acids were identified and their concentrations estimated. A linear diarylheptanoid was detected as a trace component, marking the first time this type of secondary product in Hemodoraceae has been found.

Characterization and identification of diarylheptanoids in ginger (Zingiber officinale Rosc.) using high-performance liquid chromatography/electrospray ionization mass spectrometry.

In our continuing investigation of diarylheptanoids in Zingiberaceae plants using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS), 26 diarylheptanoids were identified from fresh ginger rhizome. Of the 26 compounds, 15 diarylheptanoids appear to be new compounds. In addition, the majority of these compounds (18) were acetylated, which is different from our investigation of diarylheptanoids from turmeric, another member of the Zingiberaceae, which did not possess any acetylated diarylheptanoids. In all, five distinct groups (homologous series) of diarylheptanoids were found in extracts from ginger rhizome. These groups were differentiated by structural differences on the heptane skeletons, whereas homologs within each group differed by substitution patterns on the aromatic rings. Diagnostic fragmentation behavior in (+)- and (-)ESI-MS/MS analyses for each group of homologs, as well as information regarding polarity obtained from retention time data, allowed us to classify compounds by group and identify them based on key structural features.

Use of liquid chromatography-electrospray ionization tandem mass spectrometry to identify diarylheptanoids in turmeric (Curcuma longa L.) rhizome.

LC-ESI-MS/MS coupled to DAD analysis was used as an on-line tool for identification of diarylheptanoids in fresh turmeric rhizome extracts. Based on their mass spectra, from both negative and positive mode LC-ESI-MS/MS analysis, and supported by their DAD spectra, 19 diarylheptanoids were identified. Among these 19 compounds, curcumin, demethoxycurcumin, and bisdemethoxycurcumin were identified by comparing their chromatographic and spectral data with those of authentic standard compounds. The other diarylheptanoid compounds were identified or tentatively identified based on comparison to the three curcuminoids and each other. Twelve of the identified diarylheptanoids have not been previously reported from turmeric and six of these are new compounds.