Identification of 7,4'-dihydroxy-5-methoxyflavylium in "Dragon's blood": to be or not to be an anthocyanin

The compound 7,4'-dihydroxy-5-methoxyflavylium (dracoflavylium) was identified as the major red colorant in samples of the resin "dragon's blood", extracted from the tree Dracaena draco. The complex network of reversible chemical reactions that dracoflavylium undergoes in aqueous solution is fully described; for the first time, all the equilibrium constants that enable a complete characterisation of the system have been obtained (K'(a)=1.6 x 10(-4), K(a1)=1.0 x 10(-4), K(a2)=3.2 x 10(-8), K(Ct1)=1.0 x 10(-7), K(Ct2)=1.3 x 10(-10)). It is concluded that the red colour is due to a stable quinoid base, A, which is the major species at pH 4-7. It is further shown that this compound does not fit the commonly accepted definitions of anthocyanidin nor 3-deoxyanthocyanidin. Similarly to synthetic flavylium salts, the natural compound 7,4'-dihydroxy-5-methoxyflavylium gives rise to several species (multistate system) reversibly interconverted by external stimuli, such as pH.     

Identification and differentiation of dragon's blood in works of art using gas chromatography/mass spectrometry

Dragon's blood is a common but non-specific name for red-coloured resins that are produced by various plants, particularly exudations from plant species belonging to the genera Dracaena and Daemonorops. Although dragon's blood is mentioned in historic sources as a colourant, it has hardly ever been identified in real artworks. This paper reports the identification and discrimination of dragon's blood produced by Dracaena cinnabari, Dracaena draco as well as Daemonorops draco and Daemonorops micracantha by means of gas chromatography/mass spectrometry (GC/MS) within the context of a routine analysis of binding media used in works of art. The detection of specific flavonoid marker compounds in both underivatised and methylated methanol extracts provided the first evidence for the use of dragon's blood from all four species in various works of art from the fifteenth to nineteenth centuries. Dragon's blood was mainly used as a red colourant in gold lacquers as well as translucent glazes and paints, e.g. in reverse-glass paintings (Hinterglasmalerei).     

Raman spectroscopic analysis of dragon's blood resins-basis for distinguishing between Dracaena(Convallariaceae), Daemonorops(Palmae) and Croton(Euphorbiaceae)

"Dragon[prime or minute]s blood" is the name applied to the deep-red coloured resin obtained from various plants. The original source in Roman times, used by many cultures and esteemed for its depth of colour and mystical association, was the dragon tree Dracaena cinnabari(Convallariaceae), found only on the Indian Ocean island of Socotra, (Yemen). Additional sources emerged later, including another species of Dracaena, D. draco, from the Canary Islands and Madeira, and species in the genera Daemonorops(Palmae) from South East Asia and Croton(Euphorbiaceae) from tropical parts of both the New and Old Worlds. In this study, examples of dragon's blood resins from the Economic Botany Collections at the Royal Botanic Gardens, Kew, dating from 1851 to 1993, have been analysed non-destructively using Raman spectroscopy. The Raman spectra of well-documented, provenanced specimens have been used to establish the source of specimens of questionable or unknown origin. It has also been possible from the Raman spectra to indicate whether processing of the resins has been undertaken in the preparation of the specimens before their deposition at Kew.     

Analysis of natural red dyes (cochineal) in textiles of historical importance using HPLC and multivariate data analysis

A new analytical approach based on high-performance liquid chromatography with diode array detector (HPLC-DAD) and multivariate data analysis was applied and assessed for analyzing the red dye extracted from cochineal insects, used in precious historical textiles. The most widely used method of analysis involves quantification of specific minor compounds (markers), using HPLC-DAD. However, variation in the cochineal markers concentration, use of aggressive dye extraction methods and poor resolution of HPLC chromatograms can compromise the identification of the precise insect species used in the textiles. In this study, a soft extraction method combined with a new dye recovery treatment was developed, capable of yielding HPLC chromatograms with good resolution, for the first time, for historical cochineal-dyed textiles. After principal components analysis (PCA) and mass spectrometry (MS), it was possible to identify the cochineal species used in these textiles, in contrast to the accepted method of analysis. In order to compare both methodologies, 7 cochineal species and 63 historical cochineal insect specimens were analyzed using the two approaches, and then compared with the results for 15 historical textiles in order to assess their applicability to real complex samples. The methodology developed here was shown to provide more accurate and consistent information than the traditional method. Almost all of the historical textiles were dyed with Porphyrophora sp. insects. These results emphasize the importance of adopting the proposed methodology for future research on cochineal (and related red dyes). Mild extraction methods and HPLC-DAD/MSⁿ analysis yield distinctive profiles, which, in combination with a PCA reference database, are a powerful tool for identifying red insect dyes.     

HPLC-ESI-MS Analysis of Flavonoids Obtained from Tissue Culture of Dracaena cambodiana

Dragon's blood is a traditional medicine and used in many countries with different cultures because of its various therapeutic properties. The main bioactive constituents of dragon's blood are flavonoids, which exhibit va-rious pharmaceutical activities, such as haemostatic, analgesic, anticoagulant, and antimicrobial activities and so on, and have attracted the attention of researchers on the development of new drugs. However, the formation process of dragon's blood in nature is very slow, which cannot meet the demand of pharmaceutical uses. Dracaena cambodiana (D. cambodiana) is one of the resource plants of dragon's blood. During the course of tissue culture of D. cambodiana, 6-benzylaminopurine was added in the medium, then red secretion was discovered in the cultured medium. Twelve compounds(1―12), including 11 flavonoids, were determined via the analysis of constituents of the cultured medium by high performance liquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS) and the results were compared with that of the standards isolated from dragon's blood. Among compounds 1―12, 4,4'-dihydroxy-2-methoxydihydrochalcone(5), 4,4'-dihydroxy-2,6-dimethoxydihydrochalcone(6) and (2S)-7,3'-dihydroxy-4'-methoxyflavane(9) were major compounds of the red secretion, with contents of 15.70, 7.10 and 57.23 mg/L, respectively. Therefore, it is promising that flavonoids from dragon's blood can be obtained from the tissue culture of its resource plants for the purpose of drug development.     

A New Phenylpropanoid Glycoside from Dragon's Blood of Dracaena cambodiana

A new phenylpropanoid glycoside, named cambodianin F(1), together with three known compounds, 1-O-β-D-glucopyranosyl-2-hydroxy-4-allylbenzene(2), 1-O-(6-O-α-L-rhamnopyranosyl-β-D-glucopyranosyl)-2-hydroxy-4-allylbenzene(3) and 1,2-di-O-β-D-glucopyranosyl-4-allylbenzene(4) was isolated from the dragon's blood of Dracaena cambodiana. The new compound was elucidated by HR-ESI-MS and spectroscopic techniques(UV, IR, 1D and 2D NMR).     

Solvent effects on the thermal and photochemical reactions of 4'-iodo-8-methoxyflavylium and their consequences on the coloring phenomena caused by anthocyanins in plants

The chemistry and photochemistry of the compound 4'-iodo-8-methoxyflavylium tetrafluoroborate was studied in solvent mixtures of water and ethanol. The trans-chalcone form (Ct) is always the most stable species at equilibrium. In particular, for the first time, Ct and not the flavylium cation is reported as the most stable species in mixtures containing water at pH 1.0. The rate of Ct formation as a function of the water content exhibits a bell-shaped dependence, showing a maximum at approximately 50 % water. These trends are explained by a slower cis-trans isomerization in ethanol relative to that in water due to the expected stabilization in polar solvents of the zwitterionic intermediate; on the other hand, the decrease in the concentration of cis-chalcone (Cc) in the pseudoequilibrium with increasing water content, gives rise to a decrease in the rate of Ct formation (k(obs)=k(real)[Cc]). The hydration reaction was found to be much more efficient when water is present in low concentrations. This result has important consequences for the interpretation of the coloring phenomena caused by anthocyanins in plants, as well as for applications of flavylium compounds in the field of optical memories.     

Effect of SDS micelles on the reactivity of 4′-methoxyflavylium ion: A stopped-flow and photochemical study

The network of chemical reactions of the compound 4′-methoxyflavylium was studied in the presence of SDS micelles, using stopped-flow, UV–vis absorption and flash photolysis techniques. The results were compared with analogous experiments carried out in water. The large stabilization of the flavylium cation onto the micelles was proven to be due to an increase in the rate of the dehydration reaction, rather than a decrease of the hydration reaction; the cis–trans isomerisation of the chalcone was not affected by the micelles. It is shown that SDS micelles can be considered as an external stimulus capable of changing the system from one state to another.     

Photochromic soft materials: flavylium compounds incorporated into pluronic F-127 hydrogel matrixes

The performance of flavylium-based photochromic systems is increased by their incorporation into Pluronic F-127 matrixes, which switch from polymeric solutions to micelles to gels with changes in temperature depending on copolymer concentration. Two flavylium compounds, 7,4'-dihydroxyflavylium and 7-(N,N-diethylamino)-4-hydroxyflavylium, both exhibiting a small thermal cis-trans isomerization barrier in water were investigated. In the first system the flavylium in the gel photoswitches from the colorless trans-chalcone (Ct) species to the yellow flavylium cation (AH+) with quantum yield Phi=0.04 (25 degrees C) at pH 2.2 or to the orange quinoidal base (A) with quantum yield Phi=0.015 (25 degrees C) at pH 5.2. The photoproducts revert back to their initial form by a thermal process characterized by first-order kinetics; the rate constants exhibit a bell shape variation with pH, with a maximum at pH 4.3 (lifetime 4.2 min). The second system, 7-(N,N-diethylamino)-4-hydroxyflavylium, does not exhibit photochemistry in water but, when incorporated into the Pluronic F-127 gel, switches from yellow to red with a quantum yield of Phi=0.01 at pH 4.9. The respective thermal back reaction takes place with a lifetime of 66.7 min1. The flavylium network of chemical reactions is a good sensor for the detection of not only the critical micelle temperature but also the gelation temperature of Pluronic and like solutions and, in some instances, the exposure to UV and visible radiation.     

Development of high-performance liquid chromatographic fingerprint for the quality control of Rheum tanguticum Maxim. ex Balf

A novel, accurate and valid fingerprint method was developed by using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) to control the quality of Rheum tanguticum Maxim. ex Balf. By comprehensively comparing chromatograms of all samples, our result revealed that the chromatographic fingerprint we developed combining similarity evaluation could efficiently identify and distinguish raw materials of R. tanguticum from different sources. Besides, 21 characteristic peaks in chromatogram of R. tanguticum were unambiguously confirmed by comparison of reference compounds based on their retention time (t(R)) and UV spectra.     

In situ analysis of the Martian soil by gas chromatography: decoding of complex chromatograms of organic molecules of exobiological interest

Gas chromatography-mass spectrometry (GC-MS) will be used in future space exploration missions, in order to seek organic molecules at the surface of Mars, and especially potential chemical indicators of life. Carboxylic acids are among the most expected organic species at the surface of Mars, and they could be numerous in the analysed samples. For this reason, a chemometric method was applied to support the interpretation of chromatograms of carboxylic acid mixtures. The method is based on AutoCovariance Function (ACVF) in order to extract information on the sample--number and chemical structure of the components--and on separation performance. The procedure was applied to standard samples containing targeted compounds which are among the most expected to be present in the Martian soil: n-alkanoic and benzene dicarboxylic acids. ACVF was computed on the obtained chromatograms and plotted versus retention time: peaks of the ACVF plot can be related to specific molecular structures and are diagnostic for chemical identification of compounds.     

毛细管区带电泳法分析不同来源血竭中龙血素A和龙血素B

在系统优化了电解质溶液的pH、组成、浓度及仪器条件的基础上,建立了一种测定不同来源血竭中龙血素A和龙血素B的毛细管区带电泳(CZE)方法。采用20 kV的分离电压,25 ℃的毛细管柱温,211 nm的检测波长以及5 s的压力(3447 Pa)进样时间,在20 mmol/L的Na2B4O7缓冲溶液(用NaOH调节pH到9.98,含有10%(v/v,下同)乙腈、5.0%乙二醇和1.0%正丁醇)中,龙血素A和龙血素B在15 min内得到了有效分离与检测。方法的线性范围对于龙血素A和龙血素B分别为1.0～100.0 mg/L和0.5～100.0 mg/L。将该方法用于天然血竭及人工诱导血竭中龙血素A和龙血素B的测定,相对标准偏差在0.6%～3.8%之间,加标回收率在95.1%～105.8%之间。方法具有简单、快速、重现性较好和准确度较高的优点,可以用于血竭样品中龙血素A和龙血素B的测定。     

Synthesis and characterization of a symmetric bis(7-hydroxyflavylium) containing a methyl viologen bridge

A symmetric bis(flavylium) constituted by two 7-hydroxyflavylium moieties linked by a methylviologen bridge was synthesized. The thermodynamic and kinetics of the network of chemical reactions involving bis(flavylium) and the model compound 7-hydroxy-4'-methylflavylium was completely characterized by means of direct and reverse pH jumps (stopped flow) and flash photolysis. Both compounds follow the usual pH-dependent network of chemical reactions of flavylium derivatives. The equilibrium species of the model compound are the flavylium cation (acidic species) and the trans-chalcone (basic species) with an apparent pK'(a)=2.85. In the case of the bis(flavylium) it was possible to characterize by (1)H NMR spectroscopy three species with different degrees of isomerization: all flavylium, flavylium-trans-chalcone, and all trans-chalcone. Representation of the time-dependent mole fraction distribution of these three forms after a pH jump from equilibrated solutions of all-flavylium cation (lower pH values) to higher pH values, shows that formation of trans-chalcone is not completely stochastic (two independent isomerizations), the isomerization of one flavylium showing a small influence on the isomerization of the other. The radical of the methyl viologen bridge is formed upon reduction of the bis(trans-chalcone) with dithionite. The system is reversible after addition of an oxidant in spite of the occurrence of some decomposition.     

Cochinchin from Dracaena cochinchinensis

A new compound, Cochinchin (1), together with 7,4‘-dihydroxyflavone (2), 7-hydroxy-4‘-methoxyflavane (3),7-hydroxy-3-(4-hydroxybenzyl)chroman (4), 4‘-hydroxy-2,4-dirnethoxydihydrochalcone (5) and 4‘-hydroxy-2,4,6-trimethoxydihydrochalcone (6) was isolated from the resin (trivial name, “dragon‘s blood“) of Dracaena cochinchinensis (Lour.) S. C. Chert. The structure of 1 was elucidated on the basis of spectroscopic data as (2,3-trans)-6-allyl-2-(3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)-2,3-dihydrobenzo[1,4]dioxin which is a natural product possessing a new framework.     

GC-MS characterisation and identification of natural terpenic resins employed in works of art

Natural resins were frequently employed in the past as adhesives or as components of oleo-resinous media in paintings. The identification of vegetable resins is still an open problem. The aim of this paper is to analyse by GC-MS some vegetable resins frequently employed in paintings, such as Venice turpentine, dammar, copal, elemi, in order to identify their main components in samples both raw and aged. Some molecules are proposed as chemical markers to identify these natural resins. Two samples scraped off from XV and XVII century paintings were used to test the reliability of proposed method.     

Peak alignment using wavelet pattern matching and differential evolution

Retention time shifts badly impair qualitative or quantitative results of chemometric analyses when entire chromatographic data are used. Hence, chromatograms should be aligned to perform further analysis. Being inspired and motivated by this purpose, a practical and handy peak alignment method (alignDE) is proposed, implemented in this research for one-way chromatograms, which basically consists of five steps: (1) chromatogram lengths equalization using linear interpolation; (2) accurate peak pattern matching by continuous wavelet transform (CWT) with the Mexican Hat and Haar wavelets as its mother wavelets; (3) flexible baseline fitting utilizing penalized least squares; (4) peak clustering when gap of two peaks is smaller than a certain threshold; (5) peak alignment using differential evolution (DE) to maximize linear correlation coefficient between reference signal and signal to be aligned. This method is demonstrated with both simulated chromatograms and real chromatograms, for example, chromatograms of fungal extracts and Red Peony Root obtained by HPLC-DAD. It is implemented in R language and available as open source software to a broad range of chromatograph users (http://code.google.com/p/alignde).     

Correction of retention time shifts for chromatographic fingerprints of herbal medicines

In this study, the combination of chemometric resolution and cubic spline data interpolation was investigated as a method to correct the retention time shifts for chromatographic fingerprints of herbal medicines obtained by high-performance liquid chromatography-diode array detection (HPLC-DAD). With the help of the resolution approaches in chemometrics, it was easy to identify the purity of chromatographic peak clusters and then resolve the two-dimensional response matrix into chromatograms and spectra of pure chemical components so as to select multiple mark compounds involved in chromatographic fingerprints. With these mark components determined, the retention time shifts of chromatographic fingerprints might be then corrected effectively. After this correction, the cubic spline interpolation technique was then used to reconstruct new chromatographic fingerprints. The results in this work showed that, the purity identification of the chromatographic peak clusters together with the resolution of overlapping peaks into pure chromatograms and spectra by means of chemometric approaches could provide the sufficient chromatographic and spectral information for selecting multiple mark compounds to correct the retention time shifts. The cubic spline data interpolation technique was user-friendly to the reconstruction of new chromatographic fingerprints with correction. The successful application to the simulated and real chromatographic fingerprints of two Cortex cinnamomi, fifty Rhizoma chuanxiong, ten Radix angelicae and seventeen Herba menthae samples from different sources demonstrated the reliability and applicability of the approach investigated in this work. Pattern recognition based on principal component analysis for identifying inhomogenity in chromatographic fingerprints from real herbal medicines could further interpret it.     

Synthesis of 3-Methoxy- and 3-(β-D-Glucopyranosyloxy)flavylium Ions. Influence of the flavylium substitution pattern on the reactivity of anthocyanins in aqueous solution

The synthesis of 3-glycosyloxylated flavylium ions (anthocyanins), in particular of callistephin ( 4 ), a natural anthocyanin, is described. The structural transformations in aqueous solution and molecular complexation with chlorogenic acid ( 7 ) and caffeine ( 8 ) of the synthesized pigments 3 and 4 are investigated and compared to those of the corresponding 3-methoxyflavylium ions 1 and 2 and to those of oenin ( 5 ) and malvin ( 6 ), two very common natural anthocyanins. The results are discussed in terms of the role played by the glycosyloxy residues in the chemical properties of anthocyanins. Anthocyanin molecular complexation (copigmentaion) is quantitatively investigated by UV/VIS spectroscopy and ¹H-NMR. In particular, the UV/VIS spectroscopic data are interpreted using a general theoretical treatment, which, e.g., allows to demonstrate the formation of molecular complexes between the colourless forms of an anthocyanin and 8 .     

Applications of mass spectrometry in cultural heritage: identification of ligands employed for mordant gilding

Three different samples of gilding from wall paintings, from the Palazzo della Ragione in Padua, have been investigated by GC/MS analysis of their basic hydrolysis products obtained using trimethyl(alpha,alpha,alpha-trifluoro-m-tolyl)ammonium hydroxide (TMTFTH). The method employed allowed the identification of the mordant used for the paints, and consequently to distinguish Menabuoi's original painting from the others produced by extensive restorations. This was achieved by characterizing, among the hydrolysis products, molecular species characteristic of natural resins, siccative oils and waxes.