Characterization of Flavonoids in the Ethomedicine Fordiae Cauliflorae Radix and Its Adulterant Millettiae Pulchrae Radix by HPLC-DAD-ESI-IT-TOF-MSn

Fordiae Cauliflorae Radix (FC, the root of Fordia cauliflora Hemsl) and Millettiae Pulchrae Radix [MP, the root of Millettia pulchra (Benth.) Kurz var. laxior (Dunn) Z. Wei], which go under the same local name of “Daluosan”, have long been used in Southern China for the treatment of stroke, paralysis, dementia in children, Alzheimer’s disease and other diseases. The same local name and similar functions always confuse users. To further utilize these two ethnodrugs and identify them unambiguously, an HPLC-DAD-ESI-IT-TOF-MSn method was developed to separate and characterize the flavonoids in FC and MP. A total of 41 flavonoids were detected, of which six compounds were identified by comparing their retention time and MS data with those of the reference standards, and the others were tentatively identified based on their tandem mass spectrometry data obtained in the positive ion detection mode. Nineteen of these characterized compounds are reported from these two plants for the first time.


Introduction
Fordiae Cauliflorae Radix, the Yao medicine from Guangxi Province of China, also named Daluosan, Shuiluosan, Tugancao or Xiaxudou, originates from the root of the Leguminosae family member Fordia cauliflora Hemsl ( Figure 1A) [1][2][3]. It has been used for the treatment of stroke, paralysis, dementia in children, Alzheimer's disease, traumatic brain injury and recovery of parturients for over five hundred years. Pharmacological studies have shown that its ethanol extract can improve learning and memory ability and reverse acquired memory disorder in mice [4,5], and that it has antiaging [6], anti-inflammatory [7], hepatoprotective [8] and antioxidative [8] effects. Phytochemical studies have showed that the major constituents of FC are flavanoids, and the common chemical types are furanoflavones and pyranoflavones [2,9,10]. However, the local people often confuse FC with another Yao medicine, Millettiae Pulchrae Radix (MP), the root of Millettia pulchra (Benth.) Kurz var. laxior (Dunn) Z. Wei, which is also named Daluosan, Yulangsan, or Longyansen ( Figure 1B) [3]. It was first recorded in the Guangxi Herb Journal [9], and expresses similar activities as FC, such as for the treatment of children with infantile malnutrition, or activating blood circulation to dissipate blood stasis [3,10]. Recent studies showed that MP had a wide range of biological cardiovascular system activities [11]. It also has nootropic effects [12], is able to protect the liver [13] and has the ability to scavenge oxygen free radicals [14].
As we know, naturally derived products play an important role as a source of medicines. Ethno-medicine development is a hotspot of global drug development. The reported literatures and folk usages of FC and MP indicated that these two Yao medicines have great potential in the treatment of cardiocerebral, vascular and nervous system diseases. However, the confusion of the two medicines and the lack of reports on the global analysis of their chemical constituents, hinder the further development of both medicines. We previously quantified five flavonoids in 15 F. cauliflora samples, including root, stem and leaves, and two root samples of M. pulchra var. laxior, and tried to compare the UPLC fingerprints of the two medicines [15]. Results showed that the UPLC fingerprints of FC and MP were quite consistent within species, but distinct from each other. However, more information should be provided. For further development of FC and MP there is an urgent need to elucidate the chemical constituents of these two plants. In this paper, we choose representative samples of FC and MP, and set up a HPLC-DAD-ESI-IT-TOF-MS n method to illustrate their chemical characteristic details.

Optimization of HPLC Conditions
In order to obtain desirable HPLC chromatograms, the procedure of sample preparation was optimized in terms of the extraction solvent, extraction times of flavonoids. Four different solvents, including methanol, 80% methanol, 50% methanol and ethanol, were selected as the extraction solvents. Methanol produced the highest yield for most constituents, so it was applied as the final extraction solvent. Different columns (Merck Purospher® Star RP 18 , Agela Venusil ASB C 18 , and Dionex Acclaim® PolarAdvantage II C 18 ) were tested for the separation of the sample. By comparison, the Dionex Acclaim® PolarAdvantage II C 18 gave the best chromatographic resolution among the three columns. For the mobile phase, 0.1% (v/v) formic acid was added to improve the mass spectrometry ionization efficiency and enable symmetric peak shapes. The detection wavelength was set at 258 nm, at which most flavonoid components can be detected with greatest sensitivity. The HPLC PDA chromatograms and LC/MS base peak chromatograms (BPC) of FC and MP are given in Figure 2.

Optimization of Mass Spectrometry Conditions
Both the positive and negative ion modes were tested for the reference flavonoids. Since during our study, MS and MS n fragmentions gave more information in positive ion mode, analysis was therefore conducted in positive ion mode.

Rationale for the Characterization of Flavonoids
Known compounds in the herbal extract were identified by comparing with reference compounds according to the retention time and MS n spectra. Six peaks were identified by comparing with reference standards as pachycarin A (18), 3',4'-dimethoxy [2",3":7,8]furanoflavone (25), karanjin (29), pongaglabol (39), karanjachromene (40) and isoderricin A (41). All reference compounds exhibited [M+H] + ions of sufficient abundance in MS. The MS n spectra obtained from the reference compounds allowed us to propose the possible schemes for the fragmentation pathways of furanoflavones and pyranoflavones, and this information was used to elucidate the structure of unknown compounds.
A database (the Supplementary information- Table S1) was set up according to the reported chemicals isolated from F. cauliflora and M. pulchra var. laxior, including chemical names, structures, molecular formulae, molecular weights and so on. The elucidation procedure of unknown compounds was as follows: first of all, the molecular formulae of unknown compounds were calculated from their HRMS data, and the characteristic fragments of them were also summarized, and then the information was compared with the database. If the molecular formulae and the major fragment ions of certain compounds matched the reported chemicals in the database, their structures were elucidated. However, if the molecular formulae could not be matched with any chemicals in the database, or the molecular formulae could be matched with the database but the major fragment ions could not be matched, then they will be compared with the data retrieved in SciFinder, Dictionary of Natural Products and so on. The most plausible structure was elucidated through comprehensive analysis of MS n data. The UV spectra of the chemicals were also used to judge their structures. Meanwhile, the characteristic neutral  (Table 1). Nineteen compounds were reported from FC and MP for the first time. The tentatively identified structures and compound names are shown in Figure 3.

Identification of Furonoflavonoids
The pseudo-molecule ion [M+H] + of 3',4'-dimethoxy(2",3":7,8)furanoflavone, peak 25, in the positive ion mode was m/z 323.0909, indicating that its molecular formula was C 19 Figure 4A). It could be deduced that the dominating fragmentation pathway was retro-Diels-Alder (RDA) cleavage from the 1,3-position of the C-ring. And the 1,3 A + ion, m/z 161.02 (C 9 H 5 O 3 ) was the characteristic fragment ion of furanoflavone. The proposed fragmentation pathway can be seen in Figure 5.  The molecular formulae of compounds 10, 17, 26 and 29 were determined to be C 18 H 12 O 4 according to their HRMS data (Table 1). Compound 29 was identified as karanjin by reference [16]. Karanjin contained a 3-methoxyl moiety, and it loses a CH 3 and CH 4 in its MS 2 spectrum. In the PI MS 2 spectra of both compounds 10 and 17, the characteristic fragment ions at m/z 278.06 (predicted to be C 17 H 10 O 4 ) and m/z 176.01 (C 9 H 5 O 4 , 1,3 A + ) formed by RDA clearage suggested that the methoxyl group was link to the A-ring. According to the reported chemicals from the FC, compounds 10 and 17 were tentatively identified as O-methylpongaglabol [17] and pinnatin [18], respectively. By contrast, the fragment ions at m/z 250.06 (C 16 H 10 O 3 , [M+H-CH 3 -CO] + , base peak) and 161.02 (C 9 H 5 O 3 , 1,3 A + ) were observed in the MS 2 spectrum of compound 26, indicating that the methoxyl group was linked to the B-ring. Therefore, 26 was tentatively identified as cauliflorin A according to the reported literature [18]. were detected in compound 8. Therefore, the hydroxyl group was linked to the A-ring of compound 4, while for compound 8 was B-ring or C-ring. So they were tentatively identified as pongapinnol D [19] and pongapinnol C by comparing with the reported chemicals in the Millettia genus [18], respectively.
The molecular formulae of compounds 15 and 39 were determined to be C 17 H 10 O 4 according to their HRMS data (Table 1). Compound 39 was identified as pongaglabol by comparing with the reference compound [15]. In its PI MS 2 spectrum, the fragment ions at m/z 149.02 (C 8

Identification of Chalcones
The molecular formula of compound 11 was determined to be C 20 H 18 O 5 according to its HRMS data ( Table 1). The RDA cleavage of it at bond Y to yield the base peak ion Y A + at m/z 205.0487 (elemental composition: C 11 H 9 O 4 ) and at bond X to yield the minor ion X B + at m/z 161.0584 (elemental composition: C 10 H 9 O 2 ) could also be simultaneously detected in the MS 2 spectrum. The Y A + fragment also loses one and two methyl radicals (CH 3 ) in its MS 2 spectra ( Figure 4C) means that the A ring contains two methoxy moieties [22]. The fragmentation pathway was highly similar with what happened to flavanones. This is reasonable because cyclization of 6'-hydroxychalcones to flavanones has been reported in a number of studies demonstrating the presence of an intramolecular equilibrium between a flavanone-type and a chalcone-type molecular ion [23].
Compounds 3 and 12 were isoflavones. The predicted molecular formulae of compounds 3 and 12 were C 15 H 10 O 4 and C 16 H 12 O 4 , respectively, based on their HRMS data ( Table 1). The characteristic fragment ion at m/z 137.02 (C 7 H 5 O 3 , 1,3 A + ), which was produced after RDA cleavage from the 1,3-position of the C-ring, both existed in their MS 2 spectra ( Figure 4E) [21].

Chemical Characteristics of FC and MP
The base peak chromatograms (BPC) and PDA chromatograms of FC and MP are shown in Figure 2. In total, 41 flavanoids, including two isoflavones (two known), three pterocarpans (three known), one rotenoid, 10 chalcones (two known), 14 furanoflavones (nine known), seven pyranoflavones (four known), two flavones (one known), and two flavonones (one known) were tentatively identified, and the peak area of each compound calculated from their extracted ion chromatograms (EICs) was shown in Table 1. This is the first report of 19 chemicals from the two ethnomedicines. Some peaks were too weak to be seen clearly in the base peak chromatograms (BPCs).
Among the 41 peaks, 37 peaks were detected in FC, including 14 furanoflavones, seven pyranoflavones, eight chalcones, two isoflavones, two flavones, two flavonones and two pterocapans. Furanoflavones, pyranoflavones and chalcones are the major chemical types. However, only 15 peaks were detected in MP, including two furanoflavones, four pyranoflavones, three chalcones, two isoflavones, one rotenoid, one flavone and two pterocapans. Thus, furanoflavones were the major flavonoid chemical types in FC, while for MP the major types were chalcones and pyranoflavones. There are 11 common peaks, which are (−)-maackiain (1) (40), between FC and MP.

Identification of FC and MP
Our previous report showed that karanjin (29) was the major common peak between FC and MP, and pachycarin A (18), 3',4'-dimethoxy[2",3":7,8]furanoflavone (25), karanjachromene (40) and isoderricin A (41) can be used to differentiate between FC and MP samples [15]. However, this study indicated there were 26 compounds which were detected in FC and were not in MP, and there were four chemicals that existed in MP but not in FC (Table 1 and Figure 2C). According to the detected area of each compound (Table 1), we suggested the characteristic chemicals detected in FC, whose peak area were higher than 10 7 , including O-methylpongaglabol (10), millettocalyxin C (13), pongamol (14), pinnatin (17) However, the peak area ratio of karanjachromene (40) calculated from extracted ion chromatograms (EICs) of FC and MP was 103:1, so maybe this is the reason why karanjachromene was not detected in MP by ultra-performance liquid chromatography (UPLC) with triple-quadrupole mass spectrometry (QqQ-MS).
Besides karanjin, karanjachromene was found to possess significant antioxidant activity [29]. Few pharmacological activities were reported for pachycarin A, 3',4'-dimethoxy[2",3": 7,8]furanoflavone and isoderricin A, even though they have been known for years. However, previous researchers showed that furanoflavones can be used as antibrowning agents [30] and can also have antioxidant and radical quenching activities [31]. Pyranoflavones have antimycobacterial [32] and cytotoxic activities [33], and so on. Such information can give us clues that furanoflavones and pyranoflavones play very important roles in FC and MP. Nowadays, flavonoids are famous for their various medical efficacies, such as cardioprotective effects, antithrombotic and vasoprotective effects, antioxidation and anti-aging activies, anti-inflammatory activities [29]. Thus, the therapeutic functions of FC and MP as treatment for stroke, dementia in children and Alzheimer's disease may be due to their richness in flavonoids, but more experiments will need to be performed in order to prove this. The HPLC-DAD-ESI-IT-TOF-MS n method adopted in this study was confirmed to be a powerful method to preliminarily evaluate the ingredients in highly complex Chinese medicine extracts, especially folk medicines and other medicinal plants.
Analytical grade methanol and chromatographic grade acetonitrile were purchased from Labscan (Bangkok, Thailand), chromatographic grade formic acid was purchased from Fluka (Buchs, Switzerland). Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Plant Material
The sample of Fordia cauliflora Hemsl were collected on 12 September 2010, and identified by Professor Shou-Yang Liu (Guangxi TCM University). The root of Millettia pulchra (Benth.) Kurz var. laxior (Dunn) Z. Wei was collected and identified by professor Ren-Bin Huang (Guangxi Medical University). Voucher specimens are kept in Guangxi Botanic Garden of Medicinal Plant.

Preparation of Sample Solutions
Representative samples were ground into powder and passed through a 40 mesh sieve. Sample powder (0.2 g) was accurately weighed and transferred into a 50-mL centrifuge tube. Methanol (20 mL) was added and the mixture was sonicated at room temperature for 30 min. The extract was centrifuged at 3,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm filter and injected into the HPLC system.

Conclusions
In the present study, the HPLC-DAD-IT-TOF-MS n technique was used for rapid identification of multiple constituents in the two folk medicines, Fordiae Cauliflorae Radix and Millettiae Pulchrae Radix. As a result, a total of 41 flavonoids were successfully separated and identified, the chemical characteristics of FC and MP were elucidated respectively, resulting in the characterization of both medicines. The present study, compared with the previous studies, showed differences or improvements as follows: first of all, it is the first report of the use of the HPLC-DAD-IT-TOF-MS n method for detecting the chemical constituents in the folk medicines Fordiae Cauliflorae Radix and Millettiae Pulchrae Radix, and to characterize their chemical constituents in details. Furthermore, according to the interpretation of their mass data obtained from HPLC-DAD-IT-TOF-MS n analysis and also taking into account the data provided by the six reference standards and the established inhouse library, a total of 41 constituents were systematically characterized and identified in a single run. 41 flavanoids, including two isoflavones (two known), three pterocarpans (three known), one rotenoid, 10 chalcones (two known), 14 furanoflavones (nine known), seven pyranoflavones (four known), two flavones (one known), and two flavonones (one known) were tentatively identified. This is the first report of 19 of these chemicals from these two medicines. Thirdly, the 1,3 A + ion resulted from the RDA cleavage of C ring, m/z 161.0228 (C 9 H 5 O 3 ) was the characteristic fragment ion of furanoflavones, while the RDA cleavage 1,4 A + fragment, m/z 187.0382 (C 11 H 6 O 3 + ) was the characteristic fragment ion for pyranoflavones; which provided important clues for the identification of major flavonoids. Finally and most importantly, the two ethomedicines could be unambiguously distinguished by the results. The identification results showed that the compounds O-methylpongaglabol (10), millettocalyxin C (13), pongamol (14), pinnatin (17) (31) and isoderricin A (41) can be used to distinguish FC from MP. The results also indicated that the HPLC-DAD-ESI-IT-TOF-MS n technique is rapid and effective for structural characterization of chemical constituents in folk medicines. This work has provided comprehensive information for further quality evaluation and pharmacokinetic studies of FC and MP.