Characterization and determination of six flavonoids in the ethnomedicine “Dragon’s Blood” by UPLC-PAD-MS
© Yi et al.; licensee Chemistry Central Ltd. 2012
Received: 10 September 2012
Accepted: 8 October 2012
Published: 10 October 2012
“Dragon’s Blood” (DB) has long been used as an ethnomedicine in China to invigorate blood circulation for the treatment of traumatic injuries, blood stasis and pain. To comprehensively assess the quality of DB medicine, a precise and accurate method that can rapidly separate, characterize and quantify multiple active components of DB is crucial.
An ultra performance liquid chromatography (UPLC) coupled with photodiode array detection (PAD) and electrospray ionization mass spectrometry (ESI-MS) method was developed for characterization and determination of six flavonoids in DB. A comprehensive validation of the developed method was conducted, and confirmed that the method presented good sensitivity, precision and accuracy. All linear regressions were acquired with R2 > 0.99, and the limits of detection ranged from 0.06 to 0.83 ng. The relative standard deviation (RSD) values were found to be within the range 1.4–3.8% for the method repeatability test. Recovery studies for the quantified compounds were found to be within the range 94.2–102.8% with RSD less than 4.9%. DB samples collected from different geographical regions were analyzed by the present method, and the results demonstrated that the contents of the six flavonoids in DB samples varied significantly. Three major active components among the six flavonoids, namely dracorhodin, (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6-methylflavan-7-ol, are suggested as the index for DB quality evaluation.
Overall, the present hyphenation method is highly efficient and reliable, and hence suitable for the characterization and determination of the flavonoids of DB ethnomedicine.
KeywordsDragon's Blood Daemonorops draco UPLC-PAD-MS Characterization Quality evaluation
“Dragon’s Blood” (DB), a deep red resin secreted from the fruit of the Daemonorops draco tree, has long been used as an ethnomedicine in China to invigorate blood circulation for the treatment of traumatic injuries, blood stasis and pain [1, 2]. Flavonoids [3, 4] and resin terpenoid acids [5, 6] are the main constituents of DB. Currently, the quality evaluation for DB medicine is based on the content of only one marker compound, namely dracorhodin, which is one of the bioactive compounds identified so far in DB .
In recent years, pharmacologic studies have demonstrated that DB medicine exerts its clinical effects by inhibiting blood platelet aggregation [2, 7], and more components of DB have been found to be active in this process. For example, it is reported that the raw extract of DB dose-dependently inhibits myocardial ischemia and thrombus formation of the rats, and the coagulation time is extended significantly . (2S)-5-methoxy-6-methylflavan-7-ol, a flavonoid isolated from DB, dose-dependently inhibits aggregation of washed rabbit platelets induced by collagen, arachidonic acid and adenosine diphosphate (ADP) .
It is widely known that multiple constituents are probably involved in any herb's therapeutic functions, and that the content of a single marker compound cannot accurately reflect the quality of herbal products [10, 11]. Thus, for DB as an herbal medicine, a novel quality evaluation standard based on the contents of multiple active components is needed to comprehensively assess its quality. However, accurate means for qualitative and quantitative analysis of the multiple components simultaneously have not been reported, even though the determination of dracorhodin in DB has been carried out by TLC , HPLC  and CE , respectively. Therefore, a method for the simultaneous characterization and determination of the major active components in herbal medicines in general and DB in particular is still a top priority for accurate quality evaluation.
In the present study, a method coupling ultra-performance liquid chromatography (UPLC) with photodiode array detection (PAD) and electrospray ionization mass spectrometry (ESI-MS) was developed for the characterization and determination of six flavonoids in DB medicine. The validation results revealed that the developed method is highly efficient and reliable, and hence suitable for qualitative and quantitative analysis of DB samples. Based on the sample assay results, three major active components among the six flavonoids, namely dracorhodin, (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6- methylflavan-7-ol, are suggested as the index for quality evaluation of DB medicine.
“Dragon’s Blood” (DB) samples were collected from various regions of China. Identity of the samples was confirmed by the authors, and voucher specimens were deposited in the School of Chinese Medicine, Hong Kong Baptist University.
Reagents and chemicals
Analytical grade methanol (Labscan, Bangkok, Thailand) was used for preparation of standards and sample extraction. Chromatographic grade acetonitrile (Labscan, Bangkok, Thailand), chromatographic grade formic acid (Fluka, Buchs, Switzerland) and deionized water obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) were used for preparation of the mobile phase.
Preparation of standard and sample solutions
Linearity curves, LOD and LOQ for six compounds
y = 8580.5x + 8536.8
y = 6472.4x + 1430.2
y = 6529.5x - 2240.9
y = 25710.1x + 14943.4
y = 989.1x + 16685.5
y = 4098.2x + 14161.0
DB sample powder (0.1 g) was extracted with 10 mL of methanol by means of sonication at room temperature for 0.5 h. Sampling weight of DB is adjusted if necessary. The operations were repeated once, and the residue was washed with 4 mL of methanol. Total extracts were combined in a 25-mL volumetric flask, which was filled up to the calibration mark with extraction solvent. The extracts were then filtered through a syringe filter (0.2 μm, Alltech, Beerfield, IL, USA). An aliquot of 2 μL solution was injected for UPLC-PAD-MS analysis.
A Waters AcquityTM ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, USA) with photodiode array detection (PAD), was hyphenated to a Bruker MicrOTOFQ system by an electrospray ionization (ESI) interface (Bruker Daltonics, Bremen, Germany) for chromatographic and mass spectrometric (MS) analysis. Data analysis was conducted using DataAnalysis software version 4.0 (Bruker Daltonics). For chromatographic separation, a Waters BEH C18 column (1.7 μm, 2.1 × 100 mm) with a VanGuardTM pre-column (BEH, C18, 1.7 μm, 2.1 × 5 mm) was used. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) using a gradient program of 25% (B) in 0–2 min and 25-55% (B) in 2–15 min. The solvent flow rate was 0.3 mL/min, the column temperature was set to 40°C, and the detection wavelength was 280 nm. The conditions of MS analysis in the positive ion mode were as follows: drying gas (nitrogen), flow rate, 8 L/min; gas temperature, 180°C; scan range, 50–1600 m/z; end plate offset voltage, -500 V; capillary voltage, 4500 V; nebulizer pressure, 2.5 Bar.
Method validation and sample analysis
The calibration curve was established by plotting the peak area against the concentrations of the standards with linear regression analysis. The detection (LOD) and limit of quantitation (LOQ) of the quantified constituents were visually evaluated with a signal-to-noise ratio of about 3:1 and 10:1, respectively. Instrumental precision was investigated by repeatedly analyzing the same mixed standard solution six times, and the method repeatability was evaluated by six replicated analyses of the same DB sample. Recovery of all the quantified constituents was determined by samples at different concentration levels using a mixture of standards with 50, 100 and 200% of the quantified levels of constituents in the DB sample. All DB samples collected from various regions were analyzed using this method.
Results and discussion
Optimization of the extraction method and analysis conditions
Compared to the reflux and soxhlet extraction, sonication extraction was easier, while still satisfactory. The extraction solvent was chosen from methanol, ethanol and their various concentrations of aqueous solution. The results revealed that extraction with absolute methanol produced the highest yield for the desired analytes. Thus, methanol was chosen as the solvent for the sample extraction. Extraction times and cycles were further optimized, and the results demonstrated that exhausted extraction could be achieved when DB sample powder of 0.1 g was extracted with 10 mL of methanol by means of sonication for 0.5 h, twice.
The mass spectrometric conditions were optimized in both positive and negative ion modes; the positive ion mode was found to be more sensitive. In order to promote the formation of quasi-molecular ions [M+H] + in MS analysis, 0.1% formic acid was used in the mobile phase. MS offset voltage was further adjusted to generate characteristic fragments of the six analytes.
ESI-MS characterization of the six flavonoids
Herbal medicine is a multicomponent system with many unknown components, and the chromatographic peaks often overlap when a sample of an herbal medicine is eluted on a column. The retention time of the analytes can vary between different laboratories, while the UV spectra of the active components and other analogues are greatly similar. Thus, the direct identification of the target compounds merely based on the chromatographic parameters is still a challenge in many cases.
Compared to chromatography, mass spectrometry can provide more exclusive molecular information on the target compounds by elucidating their fragmentation pathways. These MS fragmentation characteristics greatly contribute to identify the active components from the complex mixture of herbal medicine even if the standard compounds are unavailable. Therefore, precise MS characterization of the six flavonoids is necessary in this study.
Elemental composition of major fragments in the MS spectra
Precision and accuracy study of the method
Precision (RSD %, n= 6)
Accuracy (RSD %, n= 3)
Contents of six compounds in the DB samples
Contents of six compounds (mg /g, n = 3)
4,6-Dihydroxy-2 -methoxy-3-methyldihydrochalcone (2)
(2S)-5,7- Dihydroxy-dihydroflavone (4)
(2S)-5-Methoxy flavan-7-ol (5)
Firstly, there is a significant variation in the contents of the quantified components in DB samples. Such variations may be mainly due to processing of the raw material. According to the literature , Daemonorops draco is a plant indigenous to Indonesia and Malaysia, and the resin directly collected from the fruit of Daemonorops draco tree is called raw “Dragon’s Blood” (DB). Before being sold in markets and used in clinics, the raw DB is processed to remove fruit pulp residue, and it is shaped by the addition of excipients, such as albane and dammar. However, the cost of DB is high while the cost of the excipients is low. It is hard to tell from appearance how much true DB is in any given sample sold on the market. Hence, there is an attempt to adding more excipients into the DB, and sell it for more profit. The generally low contents of all quantified constituents in some DB samples can be attributed to this reason. On the other hand, this finding confirms the necessity of developing a comprehensive quality evaluation standard based on the quantitation of the active components and on novel analysis technology to ensure the quality of DB medicine.
Secondly, comparison of the contents of the six analytes shows that the total amounts of dracorhodin (peak 1), (2S)-5-methoxyflavan-7-ol (peak 5) and (2S)-5-methoxy-6-methylflavan-7-ol (peak 6) represent about 90% of the total quantified compounds. It is undoubted that dracorhodin applicable as markers for quality evaluation of DB, due to that it is an identified bioactive component . In recent years, the anti-platelet effects of flavonoids have been confirmed by many reports in the literature [20–22], and laboratory studies suggest that “Dragon’s Blood” species exert their clinical effects by inhibiting blood platelet aggregation . The anti-platelet effect of (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6-methylflavan-7-ol have been reported [9, 23], and the underlying mechanism for anti-platelet activity of (2S)-5-methoxy-6-methylflavan-7-ol was related to inhibition of TXA2 formation via the inhibition of COX. Based on our research results and these reports, we believe that dracorhodin, (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6-methylflavan-7-ol can well represent the active flavonoids of DB to be responsible for the clinical effects of “Dragon’s Blood” against blood stasis.
Therefore, to reflect the roles of multiple compounds for the therapeutic functions, dracorhodin, (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6-methylflavan-7-ol should be chosen as analytical markers for a more comprehensive quality evaluation of DB medicine. The present hyphenation method could meet this need, making a simultaneous analysis of the three active components in a single run possible.
A UPLC-PAD-MS method was developed for the simultaneous analysis of six flavonoids in the ethnomedicine “Dragon’s Blood” (DB). The six components were characterized by online ESI-MS, and then were quantified by PAD. With respect to already existing methods, the present hyphenation procedure is highly efficient and reliable, and hence suitable for qualitative and quantitative analysis of DB samples. Based on the determination results, it is suggested that three major active components among the six flavonoids, namely dracorhodin, (2S)-5-methoxyflavan-7-ol and (2S)-5-methoxy-6- methylflavan-7-ol, be used as the index for quality evaluation of DB medicine.
Ultra performance liquid chromatography
Photodiode array detection
Thin layer chromatography
High performance liquid chromatography
This research was funded by the Faculty Research Grant of Hong Kong Baptist University (FRG2/10-11/080).
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