September 2013 Regular Article Chem. Pharm. Bull. 61(9) 941–951 (2013) 941

Simultaneous Determination of Isoflavones, Saponins and Flavones in Flos Puerariae by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Jing Lu,a Yuanyuan Xie,a Yao Tan, a Jialin Qu,a Hisashi Matsuda,b Masayuki Yoshikawa,b and Dan Yuan*,a a School of Traditional Chinese Medicine, Shenyang Pharmaceutical University; 103 Wenhua Rd., Shenyang 110016, P.R. China: and b Department of Pharmacognosy, Kyoto Pharmaceutical University; Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan. Received April 7, 2013; accepted June 6, 2013; advance publication released online June 12, 2013

An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (QTOF/MS) method is established for the rapid analysis of isoflavones, saponins and flavones in 16 samples originated from the flowers of Pueraria lobata and P. thomsonii. A total of 25 isoflavones, 13 saponins and 3 flavones were identified by co-chromatography of sample extract with authentic standards and comparing the retention time, UV spectra, characteristic molecular ions and fragment ions with those of authentic standards, or tentatively identified by MS/MS determination along with Mass Fragment software. Moreover, the method was validated for the simultaneous quantification of 29 components. The samples from two Pueraria flowers significantly differed in the quality and quantity of isoflavones, saponins and flavones, which allows the possibility of showing their chemical distinctness, and may be useful in their standardiza- tion and quality control. Dataset obtained from UPLC-MS was processed with principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the dif- ference between both Pueraria flowers. Key words Pueraria flower; isoflavone; saponin; flavone; ultra performance liquid chromatography; quadru- pole time-of-flight mass spectrometry

Pueraria lobata (WILLD.) OHWI and P. thomsonii BENTH. possess some different biological activities. For example, tec- (Leguminosae) are two commonly used medicinal plants. toridin shows potent effects on the inhibition of prostaglandin

Radix Puerariae, the roots on both plants, is a well known E2 (PGE2) production, while kakkalide has no significant herbal medicine mentioned in Chinese and Japanese Pharma- effect.17) Actually, Flos Puerariae Lobatae and Flos Puerariae copoeias,1,2) and has been used to treat common cold, influen- Thomsonii have been indiscriminate in clinical use and in the za, and wrist and shoulder stiffness, and as an antidipsotropic preparation of phytopharmaceuticals, which is a serious issue agent.3,4) On the other hand, Flos Puerariae, the flowers on in relation to the efficacy, quality control and safety. both plants, is a medicinal and edible cognate that has been Several holistic chemical profiling methods of the flowers used to ameliorate liver injury5) and relieve some symptoms of P. lobata and P. thomsonii have been reported, such as caused by excessive drinking of alcohol, such as drunkenness, quantification of 3–12 isoflavonoid glycosides and 3 saponins headache, and red face, in China, Japan and Korea.6) Many using HPLC-UV or HPLC-evaporative light scattering detec- pharmacological studies reported the extensive biological ac- tor (ELSD).9,18–20) Liu et al.21) and Shi et al.22) reported the tivities of the flower, including hepatoprotective, antioxidant, screening and identification of 13 or 18 active isoflavones hypoglycemic, and hypolipidemic activities.5,7,8) The phytoes- from ethanolic extract of P. lobata flower by using bovine trogenic isoflavonoids and saponins are the major components serum albumin (BSA) functionalized iron oxide magnetic 9–11) in the flowers as well as roots, and should be responsible nanoparticles (Fe3O4 MNPs) coupled with HPLC-MS/MS and for these aforementioned activities.12–14) 2,2′-diphenyl-1-picrylhydrazyl (DPPH) spiking HPLC-MS/ The current study is focusing on both Pueraria flowers MS. These studies demonstrate the differences in the isofla- because the phytopharmaceuticals containing the Pueraria vonoids that are present in rich amount in both flowers, i.e., flower, such as the Pueraria flower tea and drink,15) have be- kakkalide is the major component of P. lobata flower, and come popular for the treatment of alcohol intoxication and tectoridin is that of P. thomsonii flower. Concerning saponins, liver injury in China and Japan. Radix Puerariae has been only kaikasaponin III, kakkasaponin I and soyasaponin I were separately listed and recorded as Radix Puerariae Lobatae and analyzed,9,18) which possess hypoglycemic, hypolipidemic, Radix Puerariae Thomsonii in Chinese Pharmacopoeia 2005 and hepatoprotective activities.12,23) It is known that saponins and 2010 editions due to a great difference in puerarin amount are one of the important components in Pueraria sp.,24) thus, between both roots.1,16) The differences in isoflavone composi- chemical profiling and quantification of saponins are neces- tion between both Pueraria flowers consist in that kakkalide is sary for the quality control of the raw materials of Flos Puer- the major isoflavone of Flos Puerariae Lobatae, and that tec- ariae as well. toridin is that of Flos Puerariae Thomsonii. Although as phy- Generally, using conventional HPLC methods is difficult toestrogens, kakkalide and tectoridin show similar estrogenic and time-consuming for simultaneous analysis of isoflavones, effects, several studies demonstrate that both isoflavones also saponins and flavones due to the relatively low efficient sta- tionary phases. It is also one of the challenges for the accurate The authors declare no conflict of interest. quantification of the saponins possessing weak UV absorption

* To whom correspondence should be addressed. e-mail: [email protected] © 2013 The Pharmaceutical Society of Japan 942 Vol. 61, No. 9 using a UV detector. Compared to HPLC, ultra performance Shenyang Pharmaceutical University. liquid chromatography (UPLC) can provide a high peak ca- Instrumentation and Chromatographic Conditions pacity, greater resolution, increased sensitivity, and higher UPLC analysis was performed on a Waters ACQUITY speed of analysis. For example, Wu et al. simultaneously iden- HSSC18 column (2.1×100 mm, 1.8 µm, Waters, Milford, MA, tified 92 compounds, including steroidal saponins, homoisofla- U.S.A.) at 40°C. The mobile phase consisted of (A) water con- vanones and other components in Sheng-Mai San, a traditional taining 0.2% formic acid and (B) acetonitrile containing 0.2% Chinese medical formula, within 14 min by UPLC-high defi- formic acid with gradient elution (linear gradient from 5% B nition mass spectrometry (HDMS).25) TOF/MS can provide to 15% B in 1 min, followed by linear gradient from 15% B accurate molecular formula for unknown compounds, which to 45% B between 1 and 7 min, and linear gradient from 45% is very helpful for structural identification. Chu et al. charac- B to 50% B between 7 and 9 min, finally a 9–10 min linear terized 22 astragalosides from the methanolic extract of Radix gradient from 50% B to 65% B). Re-equilibration duration Astragali using quadrupole time-of-flight mass spectrometry was 2 min between individual runs. The flow rate was kept (QTOF/MS) method.26) Since many unknown saponins may at 0.6 mL/min and 2 µL of standard and sample solution were be present in the Pueraria flower and most are new or without injected in each run. reference standards, using TOF/MS to identify them is very Identification of marker compounds by UPLC-MS was per- important based on the molecular formula and fragmentation formed on Waters QTOF Xevo G2 (Waters Corp., Manchester, of the known saponins. U.K.) equipped with an electrospray ionization (ESI) source, In the present study, we developed a UPLC-QTOF/MS which gives a resolution of 10000 (FWHM) and mass accu- method for the simultaneous analysis of isoflavones, saponins racy error less than 5 ppm. Leucine-enkephalin was used as and flavones for the evaluation of the chemical consistency the lock mass to generate an [M−H]− ion (m/z 554.2615) in between in the flowers of P. lobata and P. thomsonii. the LockSpray mode at a concentration of 50 pg/µL at an infu- sion flow rate of 10µ L/min. The ESI source was operated in Experimental negative ionization mode with the capillary voltage at 2.5 kV, Chemicals and Reagents Thirty-one reference com- and the cone voltage was set to 40 V. Source and desolvation pounds were used in the present study, and the chemical temperatures were set at 130 and 450°C, respectively. The structures were shown in Fig. 1. Puerarin (1), daidzin (5), cone and nebulization gas flows were 50 and 800 L/h, respec- daidzein (16) and luteolin (18) were purchased from Funa- tively. All data collected in centroid mode were acquired using koshi Co., Ltd. (Tokyo, Japan), and glycitin (6) was from Masslynx™ NT 4.1 software (Waters Corp., Milford, MA, Shanghai Forever Biotech Co., Ltd. (Shanghai, China). Other U.S.A.). used reference compounds were isolated from the extracts Two different MS scanning experiments were used. (1) MSE of the flowers of P. lobata and P. thomsonii in our previous experiment (E represents collision energy) uses an intelligent studies.9,10,13,27) They included 6-hydroxygenistein-6, 7-di-O- approach where parallel alternating scans are acquired both at glucoside (2), 6-hydroxygenistein-7-O-glucoside (7), rutin (8), low-collision and high-collision energy to obtain precursor ion tectorigenin-7-O-xylosylglucoside (10), genistin (11), tectoridin information and full-scan mass fragment with precursor ion (12), 6-hydroxybiochanin A-6, 7-di-O-glucoside (13), gehuain information in a single analytical run, respectively. The MSE (14), glycitein (17), kakkalide (20), genistein (22), tectorigenin experiment in two scan functions was carried out as follows. (25), biochanin A (27), astragaloside VIII (28), soyasaponin I Function 1: m/z 100–1000, 0.5 s scan time, 0.02 s inter-scan (29), irisolidone (30), soyasaponin IV (31), kaikasaponin III delay, 6 V collision energy, and function 2: m/z 100–1000, 0.5 s (32), kaikasaponin II (33), kaikasaponin I (34), kakkasaponin scan time, 0.02 s inter-scan delay, collision energy ramp of I (35), azukisaponin I (36), baptisiasaponin I (37), phaseoside 50–60 V. (2) MS/MS experiments were carried out by ramping IV (38), kakkasaponin II (39) and kakkasaponin III (41). The collision energies from 50 and 60 V. identity of these compounds were confirmed by melting point, Preparation of Standard Solutions Twenty-nine refer- UV, IR, 1H- and 13C-NMR and MS, and their purities evalu- ence compounds, including 6-hydroxygenistein-6,7-di-O- ated with HPLC-photodiode array detector (PDA) were more glucoside (peak 2), glycitin (peak 6), 6-hydroxygenistein- than 95%. Acetonitrile (ACN, HPLC-MS grade) and formic 7-O-glucoside (peak 7), rutin (peak 8), tectorigenin-7-O- acid (spectroscopy grade) were purchased from Fisher Scien- xylosylglucoside (peak 10), genistin (peak 11), tectoridin tific U.K. (Loughborough, U.K.). Ultrapure water (18.2 MΩ) (peak 12), 6-hydroxybiochanin A-6, 7-di-O-glucoside (peak daily prepared with a Milli-Q water purification system (Mil- 13), gehuain (peak 14), daidzein (peak 16), glycitein (peak lipore, Bedford, MA, U.S.A.). Leucine-enkephalin was ob- 17), luteolin (18), kakkalide (peak 20), genistein (peak 22), tained from Sigma-Aldrich (St. Louis, MO, U.S.A.). tectorigenin (peak 25), biochanin A (peak 27), astragaloside Plant Materials Sixteen samples (samples #1–#16) were VIII (peak 28), soyasaponin I (peak 29), irisolidone (peak directly obtained from China or Japan, which were collected 30), soyasaponin IV (peak 31), kaikasaponin III (peak 32), in the bud between June and October (Table 1). The plant kaikasaponin II (peak 33), kaikasaponin I (peak 34), kakkasa- materials were authenticated by Professor Weining Wang ponin I (peak 35), azukisaponin I (peak 36), baptisiasaponin (Liaoning Institute for Food and Drug Control, China). They I (peak 37), phaseoside IV (peak 38), kakkasaponin II (peak were divided into P. lobata or P. thomsonii according to the 39) and kakkasaponin III (peak 41), were accurately weighed, shape of bractlet, length of the calyx, proportion of the calyx- and dissolved in MeOH to give individual stock solutions at a teeth to calyx-tube, the presence or absence of callosum at final concentration of 0.2 mg/mL. Series of working standard the base of vexillum, and both of angle and proportion of two solutions were prepared by appropriate dilution of the stock 28,29) claws at the base of each wing. The voucher specimens solution with 80% MeOH–H2O in order to prepare calibrators. are kept in the reference library for the medicinal herbs in All solutions were stored at 4°C in refrigerator before analysis. September 2013 943

Fig. 1. Structures of 31 Reference Compounds Identified from P. lobata and P. thomsonii. Glc, Glucose; Xyl, Xylose; Rha, Rhamnose; Gal, Galactose

Sample Preparation Powdered herbal materials (0.1 g, injecting each solution in triplicates, and then the calibration passed through a 500 µm mesh sieve) were ultrasonically dou- curves were constructed by plotting the peak area versus the ble-extracted with 10 mL of MeOH–H2O (60 : 40) for 30 min. concentration of each analyte. The mixtures were centrifuged at 3000 rpm for 10 min, and Limit of Detection (LOD) and Limit of Quantitation the supernatants were diluted to 50 mL with methanol in (LOQ) The stock solutions of 29 reference compounds were a volumetric flask. An aliquot of each µ2 L filtrate filtered diluted to a range from 0.29 to 20.0×10−3 µg/mL, and the in- through a 0.22 µm PTFE syringe filter (Whatman, MN, Nal- jection volume was 2 µL. The LOD and LOQ were determined gene, Advantec) was injected into the UPLC instrument for at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. analysis. Precision, Accuracy and Repeatability The intra- and Method Validation for Quantification Among the inter-day precisions were evaluated by analyzing known con- 41 identified compounds, 15 isoflavones, 12 saponins and centrations of the 8 analytes, including 3 isoflavones tectoridin 2 flavones were quantified on an UPLC-QTOF/MS using (12), kakkalide (20) and irisolidone (30), 3 saponins sayasapo- quasi-molecular ion chromatograms (XICs, extracted ion nin I (29), kaikasaponin III (32) and phaseoside IV (38), and chromatograms, with a 0.02 Da window), which peak area was 2 flavones rutin 8( ) and luteolin (18) in six replicates during a integrated at the expected retention times under full-scan MS single day and by duplicating the experiments on 3 successive conditions (Waters QuanLynx™ version 4.0 software). days. Six different sample solutions prepared from the same Calibration Curves Calibration curves (5-point) were sample were analyzed to confirm the repeatability of the de- obtained using external standard calibrations for 29 analytes veloped assay. Stability of sample solutions was analyzed at 0, 944 Vol. 61, No. 9

Table 1. Herbal Materials Samples Used in the Study

Sample No. Authentication (purchased from) Collection area/date Voucher No.

1 P. lobata Dandong, China/Aug. 2006 A0270608C 2 〃 Toyama, Japan/Sept. 2006 A0270609D 3 〃 Dandong, China/Aug. 2006 A0270610O 4 〃 Dalian, China/Aug. 2006 A0270611P 5 P. thomosonii (Kanebo Co., Ltd.) Guangxi, China/Sept. 2006 A0270310A 6 〃 (Hangzhou Botanical Tech Co., Ltd.) Guangxi, China/Aug. 2006 A0270608B 7 〃 (Hangzhou Botanical Tech Co., Ltd.) Guangxi, China/Oct. 2006 A0270610E 8 〃 (Hangzhou Botanical Tech Co., Ltd.) Hunan, China/Sept.2006 A0270609F 9 〃 (Hangzhou Botanical Tech Co., Ltd.) Hunan, China/Aug. 2006 A0270610G 10 〃 (Changan TCM Co., Ltd.) Liaoning, China/ Oct. 2012 A0270704H 11 〃 (Bright Decoction Pieces Co., Ltd.) Guangxi, China/Oct. 2012 A0271210I 12 〃 (Imperial Jindan Pharmaceutical) Liaoning/June 2012 A0271206J 13 〃 (Morality Thatched Cottage Pharmacy) Hunan, China/Aug.2012 A0271208K 14 〃 (Four-Sided Pharmacy) Hubei, China/July 2012 A0271200L 15 〃 (National Unity Pharmacy) Anhui, China/July 2012 A0271200M 16 〃 (Beneficial Cottage Pharmacy) Guangzhou, China/ July 2012 A0271200N

2, 4, 8, 12 and 24 h at room temperature, respectively. Varia- gradient elution as described in chromatographic conditions tions were expressed by relative standard deviations (RSD). of Experimental section, the 31 standards were satisfactorily The recovery was used to evaluate the accuracy of the separated within 10 min. method. A known amount of the 8 standards mixed solutions Mass Spectrometry Analysis of 41 Marker Compounds were added into a certain amount of the P. thomsonii flower from the Flowers of P. lobata and P. thomsonii In order (0.1 g, sample #8). The mixture was extracted and analyzed to characterize the chemical composition, the methanolic using the method mentioned above. Three replicates were per- extracts of the flowers of P. lobata (sample #1) and P. thom- formed for the test. sonii (sample #8) were subjected to UPLC-ESI/MS analysis. Chemometric Data Analysis The UPLC-MS data of P. Forty-one specific peaks (labeled peaks 1–41, Fig. 3) in the lobata and P. thomsonii samples were analyzed by Marker- UPLC chromatograms were characterized by typical UV Lynx XS software (Waters, Manchester, U.K.). The param- absorptions obtained with Waters PDA detector, with the eters were set as following: retention time range 1.0–10.5 min; maximum absorptions displaying at 259–265 nm for isofla- mass range m/z 100–1000 Da; retention time tolerance 0.1 min; vones, 263–293 nm and 357–362 nm for flavones, and without mass tolerance 0.05 Da; width of an average peak at 5% height an obvious UV absorption at more than 230 nm for saponins and peak-to-peak baseline noise were automatically calcu- or other compounds. By co-chromatography of sample extract lated; marker intensity threshold 10.0; noise elimination level with authentic standards and comparing the retention time, 6.0; and isotopic peaks were excluded for analysis. UV spectra, characteristic molecular ions and fragment ions (Table 2 and Supplemental Data S3) with those of authentic Results and Discussion standards that were isolated in our previous studies, the com- Optimization of Extraction Procedure In order to pounds corresponding to 31 peaks were identified as puerarin obtain satisfactory extraction efficiency for all the analytes, (peak 1), 6-hydroxygenistein-6,7-di-O-glucoside (peak 2), extractive methods (ultrasonic and refluxing), solvents (40%, daidzin (peak 5), glycitin (peak 6), 6-hydroxygenistein-7-O- 60% and 80% methanol), and time (15 and 30 min) were as- glucoside (peak 7), rutin (peak 8), tectorigenin-7-O-xylosyl- sessed based on single factor experiments. The best extraction glucoside (peak 10), genistin (peak 11), tectoridin (peak 12), efficiency was obtained by sonication extraction with 60% 6-hydroxybiochanin A-6, 7-di-O-glucoside (peak 13), gehuain methanol for 30 min. (peak 14), daidzein (peak 16), glycitein (peak 17), luteolin Optimization of Chromatographic Separation A mixed (18), kakkalide (peak 20), genistein (peak 22), tectorigenin solution of 17 isoflavones, 12 saponins, 2 flavones and the (peak 25), biochanin A (peak 27), astragaloside VIII (peak methanolic extracts of P. lobata (sample #1) and P. thomsonii 28), soyasaponin I (peak 29), irisolidone (peak 30), soyasa- (sample #8) were used for the optimization of UPLC condi- ponin IV (peak 31), kaikasaponin III (peak 32), kaikasaponin tions, the representative UPLC-UV and UPLC-MS chromato- II (peak 33), kaikasaponin I (peak 34), kakkasaponin I (peak grams of which are presented in Fig. 3. The chromatographic 35), azukisaponin I (peak 36), baptisiasaponin I (peak 37), behavior varied with the concentrations of formic acid (0.0%, phaseoside IV (peak 38), kakkasaponin II (peak 39) and kak-

0.1% and 0.2%) and different columns (HSSC18, 2.1×100 mm, kasaponin III (peak 41). 1.8 µm, Waters; ethylene bridged hybrid (BEH), 2.1×100 mm, Due to absence of reference compounds, the compounds 1.7 µm, Waters). The results obtained showed that the sepa- corresponding to the rest 10 peaks were tentatively identi- rations on the HSSC18 column using the gradient solvent fied by MS/MS determination along with Waters MassFrag- systems, consisting of water (containing 0.2% formic acid, ment software, the characteristic UV absorption spectra, solvent A) and ACN (containing 0.2% formic acid, solvent B), and comparison with literature data (see Fig. 2, Table 2 and were better than other tested conditions. Using the optimal Supplemental Data S3). Glycitein-O-pentosyl-hexoside (peak September 2013 945

Table 2. Marker Compounds Identified from the Flowers of P. lobata and P. thomsonii by UPLC-QTOF/MS Methods

Aglycones or t UV [M−H]− Comparison Peaks No. Compounds R diagnostics (min) (nm) (m/z) with standards fragments (m/z)

1 Puerarin 1.55 262 415.1028 253, 223 yes 2 6-Hydroxygenistein-6,7-di-O-glucoside 1.61 263 609.1451 447, 285 yes 3 Dihydrokaempferol-O-hexoside 1.71 293 449.1076 287, 269 no 4 Glycitein-O-pentosyl-hexoside 1.82 262 577.1542 445, 283, 268, 240 no 5 Daidzin 1.88 260 415.1022 253 yes 6 Glycitin 1.99 262 445.1128 283, 268, 239 yes 7 6-Hydroxygenistein-7-O-glucoside 2.01 263 447.0952 285, 267 yes 8 Rutin 2.17 263, 357 609.1455 301, 285 yes 9 Genistein-O-pentosyl-hexoside 2.23 259 563.1405 431, 269 no 10 Tectorigenin-7-O-xylosylglucoside 2.33 264 593.1513 299, 284, 255 yes 11 Genistin 2.48 259 431.0974 269 yes 12 Tectorodin 2.60 264 461.1079 299, 284, 255 yes 13 6-Hydroxybiochanin A-6,7-di-O-glucoside 3.21 260 623.1605 461, 299, 284 yes 14 Gehuain 3.26 259 591.1725 297, 282, 267 yes 15 Isomer of tectorigenin 3.28 261 299.0554 284 no 16 Daidzein 3.61 260 253.0500 223 yes 17 Glycitein 3.88 262 283.0599 268 yes 18 Luteolin 3.92 263, 362 285.0400 133 yes 19 Biochanin A-O-pentosyl-hexoside 4.13 259 577.1553 445, 283, 268 no 20 Kakkalide 4.19 265 607.1666 313, 298, 283 yes 21 Isomer of kakkalide 4.31 260 607.1669 313, 298, 283 no 22 Genistein 4.61 259 269.0450 133 yes 23 Biochanin A-O-hexoside 4.64 259 445.1120 283, 268 no 24 Irisolidone-O-hexoside 4.72 265 475.1245 313 no 25 Tectorigenin 4.80 264 299.0557 284, 255 yes 26 Isomer of irisolidone 5.20 260 313.0708 298, 283, 255 no 27 Biochanin A 7.10 259 283.0604 268 yes 28 Astragaloside VIII 7.18 n.d. 911.4998 765, 615, 457 yes 29 Soyasaponin I 7.22 n.d. 941.5096 795, 615, 457 yes 30 Irisolidone 7.32 265 313.0713 298, 283, 255 yes 31 Soyasaponin IV 7.44 n.d. 765.4436 615, 457 yes 32 Kaikasaponin III 7.59 n.d. 925.5161 779, 617, 599, 441 yes 33 Kaikasaponin II 7.72 n.d. 925.5161 779, 617, 599, 441 yes 34 Kaikasaponin I 8.06 n.d. 779.4582 617, 599, 441 yes 35 Kakkasaponin I 8.16 n.d. 895.5042 749, 599, 441 yes 36 Azukisaponin I 8.17 n.d. 779.4590 617, 441 yes 37 Baptisiasaponin I 8.28 n.d. 895.5062 749, 599, 441 yes 38 Phaseoside IV 8.47 n.d. 923.4995 777, 597, 439 yes 39 Kakkasaponin II 9.03 n.d. 777.4427 597, 439 yes 40 Abrisapogenol F-O-rhamnosyl-pentosyl-glucuronide 9.19 n.d. 893.4900 747, 597, 439 no 41 Kakkasaponin III 9.36 n.d. 893.4900 747, 597, 439 yes

tR, the retention time of the detected peaks. n.d., not detectable. The details of spectroscopic data were shown in Supplemental Data S3.

4), abrisapogenol F-O-rhamnosyl-pentosyl-glucuronide (peak together with inquiring the literatures about the isoflavonoids 40) and dihydrokaempferol-O-hexoside (peak 3) are three found in the flowers of P. thunbergiana, the compound cor- representatives for the structural identification of isoflavone, responding to peak 4 was tentatively identified as glycitein-O- saponin and flavone from the Pueraria flower extracts in the pentosyl-hexoside.30)

UPLC-QTOF-MS/MS determination. Peak 40 (tR 9.19 min) and kakkasaponin III (peak 41, tR Peak 4 was present in the UPLC chromatogram of the ex- 9.36 min) were both found in the extract of P. lobata flower tract of P. thomsonii flower (sample #8), and showed a typical (sample #1). Both peaks separately generated the [M−H]− ions isoflavone UV absorption at 262 nm, a negative molecular ion at m/z 893.4900, corresponding to the same molecular formula − at m/z 577.1542 [M−H] corresponding to the molecular for- C47H73O16. As shown in Supplemental Data S3, high colli- mula C27H29O14, and two fragment ions at m/z 445.1148 (loss sion energy resulted in abundant fragment ions, such as m/z of a pentosyl, 132 mass units), 283.0601 (loss of a hexosyl and 747.4323 (loss of a rhamnosyl, 146 mass units), 597.3793 (loss a pentosyl, 294 mass units), suggesting that it is an isoflavone of a rhamnosyl, a pentosyl and H2O, 296 mass units), 439.3601 diglycoside. According to its same UV absorbance spectrum of the aglycone ion (loss of a rhamnosyl, a pentosyl and gluc- as that of glycitin, and the major fragment ions of glycitein, uronyl, 454 mass units) (see Fig. 4). These fragment ions were 946 Vol. 61, No. 9

Fig. 2. Structures of 7 Tentatively Identified Isoflavones, Saponins and Flavones from P. lobata and P. thomsonii

‘Ra’ represents the linkage was not determined. pent, pentose; hex, hexose; rha, rhamnose. The rest 3 tentatively identified isoflavones were not shown, which are the isomers of known compounds. also shown in the fragmentation pathways as kakkasaponin peaks 35 and 36 that were separately characterized and quan- III. Until now, only sapogenin abrisapogenol F yields proton- tified according to their different quasi-molecular ions using ated ion m/z 439.3576 among the saponins that have been re- extracted ion chromatograms (XICs, with a 0.02 Da window) ported to be found in the medicinal Pueraria sp. plants. Thus, under full-scan MS conditions. the compound corresponding to peak 40, an isomer of and Method Validation Good liner calibration curves were kakkasaponin III, was tentatively identified as abrisapogenol obtained with 29 tested reference standards (R>0.995, see F-O-rhamnosyl-pentosyl-glucuronide. Supplemental Data 1). Due to low content or without reference Peak 3 was present in the UPLC chromatogram of the ex- standards, the calibrations for puerarin, daidzin and ten tenta- tract of P. thomsonii flower (sample #8). It showed a typical tively identified compounds were not established. Because of dihydroflavone UV absorption at 293 nm, a negative molecular a great difference in the contents of rutin, tectorigenin-7-O- ion at m/z 449.1076 corresponding to the molecular formula xylosylglucoside, tectoridin, luteolin, kakkalide, tectorigenin,

C21H21O11, and two fragment ions at m/z 287.0558 (loss of a biochanin A, irisolidone, kakkasaponin I, baptisiasaponin I hexosyl, 162 mass units) and m/z 269.0443 (loss of a hexosyl and phaseoside IV between the herbal materials, two calibra- and a H2O, 180 mass units), suggesting that it should be a di- tions were established to serve for the low and high content hydroflavone glycoside. The compound corresponding to peak levels. The LODs and LOQs were in the range from 0.29 to 3 possessed the same UV absorbance spectrum, molecular 20.0×10−3 µg/mL and from 0.96 to 66.8×10−3 µg/mL, respec- mass, and two major fragment ions as those of dihydrokaemp- tively. ferol-glucoside,31) which is the only one dihydroflavonoid with The precision and recovery tests were done for 8 major the molecular formula C21H21O11 found in the literatures on the marker compounds by the same methods described in method dihydroflavonoid compounds from Leguminosae plants, thus, validation for quantification of Experiment section. This meth- it was tentatively identified as dihydrokaempferol-O-hexoside. od exhibited good reproducibility with intra- and inter-day Using the similar methods described above, 8 isoflavones variations (RSD) of less than 4.1% (see Supplemental Data 2). (peaks 4, 9, 15, 19, 21, 23, 24 and 26), 1 saponin (peak 40) The recovery for these markers ranged from 96.2% to 106.9%, and 1 dihydroflavone (peak 3) were tentatively identified, with RSD ranging from 2.7% to 7.9% (see Supplemental Data as listed in Table 2 (more spectroscopic data was shown in 2). Thus, this analytical procedure is accurate and sufficiently Supplemental Data S3). For the seven glycosidic compounds sensitive for the simultaneous quantification of the major iso- (3, 4, 9, 19, 23, 24 and 40), however, sugar anomeric configu- flavones, saponins and flavones in both Pueraria flowers. ration and substituted position cannot be confirmed only based Quantification of Isoflavones, Saponins and Flavones on their MS spectra. The identified or tentatively identified in the Flowers of P. lobata and P. thomsonii Chemical compounds were satisfactorily separated under the UPLC- profiling and quantification of isoflavones, saponins and fla- PDA and/or UPLC-MS conditions, except for peaks 28 and 29, vones from the flowers of P. lobata and P. thomsonii using September 2013 947

Fig. 3. The Representative UPLC-UV and BPI Chromatograms of Mixed Reference Compounds (a and d) and the Extracts of the Flowers of P. lobata (Sample # 1, b and e) and P. thomsonii (Sample # 8, c and f): (a–c) UPLC Chromatograms Detected at 254 Nm; (d–f) BPI (Base Peak Intensity) Chromatograms in Negative Ionization Mode See Table 2 for the peak numbers, and see Experimental section for UPLC-QTOF/MS conditions.

Fig. 4. Fragmentation Pattern (a) and MS and MSE Spectra (b and c) of Abrisapogenol F-O-Rhamnosyl-Pentosyl-Glucuronide (Peak 40) 948 Vol. 61, No. 9 18 0.08 0.08 0.31 0.22 0.42 0.22 0.19 0.07 0.05 0.05 0.06 0.05 0.07 0.04 0.08 0.06 8 tr. tr. tr. Flavones 1.50 3.51 0.17 0.85 0.91 4.64 1.23 0.36 0.77 0.27 0.09 0.36 0.13 9.70 9.07 Ts 25.0 33.0 13.4 23.1 40.7 31.1 42.4 34.8 38.6 10.8 23.2 12.2 10.3 60.6 tr. tr. tr. tr. tr. tr. tr. tr. tr. 41 n.d. 0.31 1.19 0.63 0.81 0.84 0.39 39 0.40 0.08 0.24 0.73 0.2 0.28 0.42 0.23 0.12 0.14 0.13 0.08 0.09 0.28 0.15 0.28 38 1.10 1.98 0.72 1.72 2.74 1.03 2.43 1.89 2.43 0.53 1.19 1.09 0.39 6.15 0.35 0.35 37 1.47 2.02 3.51 2.08 2.77 3.21 2.34 0.47 0.56 1.53 0.09 1.01 1.42 0.86 1.38 1.27 tr. tr. 36 0.15 0.44 0.23 0.20 0.53 0.20 0.11 0.12 0.1 0.33 0.1 0.10 0.30 0.09 1.9 5.47 2.16 3.96 2.96 3.27 3.61 5.31 5.08 6.14 8.23 2.12 2.10 1.43 1.78 35 12.30 Saponins 34 0.70 0.16 0.62 2.08 0.3 1.12 1.58 0.78 0.21 0.26 0.12 0.17 0.74 0.31 0.42 0.74 tr. tr. tr. tr. tr. tr. 33 1.50 1.28 1.36 0.68 2.96 1.43 4.37 4.86 1.63 5.74 8.55 2.81 7.27 5.85 6.14 2.13 4.45 3.63 1.26 1.42 1.43 32 11.5 11.4 12.6 14.2 15.2 31 n.d. 0.50 0.09 1.31 0.95 0.67 1.25 0.64 0.07 0.06 0.46 0.15 0.86 0.08 0.43 0.72 5.53 3.57 9.31 6.97 9.79 9.69 3.39 7.02 3.69 2.66 3.12 2.65 29 14.7 10.6 16.3 19.1 28 0.43 0.29 0.15 0.11 0.18 0.46 0.84 0.16 0.17 0.29 0.34 0.23 0.22 0.62 0.23 0.18 Ti 37.0 40.5 81.3 56.6 81.6 85.7 22.2 18.1 32.5 19.4 30.4 63.7 24.5 34.7 91.2 112 tr. tr. 30 n.d. n.d. n.d. n.d. 0.20 0.03 0.36 0.03 0.22 0.06 3.45 4.95 4.11 2.76 tr. tr. tr. tr. tr. tr. 27 0.05 0.04 0.01 0.02 0.01 1.04 1.42 1.02 0.01 0.94 P. lobata and thomosonii tr. tr. tr. tr. 25 6.45 0.95 1.05 7.01 4.28 4.02 9.11 0.56 0.27 1.38 0.56 15.0 tr. tr. tr. tr. 22 1.40 0.37 0.51 0.27 1.02 1.32 0.73 1.72 0.22 0.27 0.22 0.16 0.78 0.81 0.38 4.28 3.06 0.32 0.13 0.21 0.10 0.90 0.35 tr. 20 14.9 10.3 13.1 17.7 0.55 9.37 1.32 1.26 0.57 0.52 tr. tr. tr. tr. tr. tr. tr. tr. 17 10.6 12.4 tr. tr. tr. 16 0.02 0.17 0.05 0.10 0.04 0.14 0.04 0.21 0.08 0.06 0.03 0.06 0.04 tr. tr. 14 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.19 0.18 0.22 0.35 Isoflavones tr. tr. tr. tr. tr. tr. tr. 13 n.d. n.d. n.d. n.d. n.d. n.d. 8.77 5.81 9.89 P. lobata , and the rest samples are as thomosonii . Ti and Ts represent the sum quantities of 15 isoflavones and 12 saponins, respectively. n.d., not detectabletr., trace of detection). ( < limit tr. tr. tr. tr. 12 9.37 5.47 5.74 4.89 3.59 5.52 5.68 23.9 14.1 36.0 42.7 32.6 tr. 11 0.58 1.68 1.27 1.00 0.96 1.17 0.86 0.25 0.13 0.36 0.48 0.12 0.45 0.32 0.40 2.31 1.30 1.06 2.57 10 15.0 28.3 37.6 27.6 28.5 25.5 44.9 25.8 24.9 22.7 26.9 26.6 7 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.71 0.40 0.28 0.36 0.27 0.44 0.69 6 n.d. n.d. n.d. n.d. 0.53 0.49 3.44 3.70 0.51 2.15 2.52 6.24 0.89 2.04 0.34 0.21 2 0.71 tr. tr. tr. tr. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 18 18.2 17.8 9 8 7 6 5 4 3 2 1 The samples #1–#4 are botanically identified as 12 11 10 16 15 13 14 ple No. Sam - Table 3. Content (mg/g) of 15 Isoflavones, 12 Saponins, and 2 Flavones in the Flowers of minimum of the linear range), as described in Experimental section of Method Validation. The numbers in bold represent the compounds as described in Fig. 1. the compounds as described The numbers in bold represent of Method Validation. section in Experimental range), as described of the linear but < minimum of detection ( > limit September 2013 949

Fig. 5. PCA Scores Plot (A), OPLS-DA Plot (B) and S-Plot (C) of 4 P. lobata and 12 P. thomosonii Samples Circles and open triangles in (A) and (B) represent P. lobata and P. thomosonii, respectively. The points a–f in the S-plot (C) represent the top six leading markers. the UPLC-QTOF/MS method were carried out (see Fig. 3 and for glycitin, 0.32–1.68 mg/g for genistin, and 0.16–1.72 mg/g Table 3). There is a significant difference in the chemical pro- for genistein). However, daizin and daizein, two important filing patterns between both flowers. soy-isoflavones, were detected in trace or small amount in Kakkalide and irisolidone were detected in richer abun- both flowers. Total content of 15 isoflavones was much higher dance in P. lobata flower (10.3–17.7 mg/g for kakkalide, and in the P. thomsonii flower (30.4–112.6 mg/g) than in the P. lo- 2.76–4.95 mg/g for irisolidone) than in P. thomsonii flower bata flower (18.1–24.5 mg/g). (trace—4.28 mg/g for kakkalide, and 0.00–0.36 mg/g for Although Pueraria root and flower are both botanically irisolidone). On the other hand, tectorigenin-7-O-xylosylglu- from P. lobata and P. thomsonii, their isoflavone composition coside, tectoridin and tectorigenin were detected in richer is quite different from each other. According to Du et al.’s abundance in the P. thomsonii flower (15.0–44.9 mg/g for study,32) we make a comparison of isoflavone composition be- tectorigenin-7-O-xylosylglucoside, 3.59–42.7 mg/g for tectori- tween Pueraria roots and Pueraria flowers as follows. Opposite din, and 0.27–15.0 mg/g for tectorigenin) than in the P. lobata to flowers, the total content of 13 isoflavones in P. lobata root flower (1.06–2.57 mg/g for tectorigenin-7-O-xylosylglucoside, (53.6–134.7 mg/g) was much higher than that in P. thomsonii and trace for tectoridin and tectorigenin). Thus kakkalide root (5.36–12.5 mg/g).32) Among them, puerarin (21.1–54.3 mg/g and irisolidone are the major isoflavones of P. lobata flower, in P. lobata root and 2.23–9.27 mg/g in P. thomsonii root) and while tectorigenin-7-O-xylosylglucoside, tectoridin and tec- daidzin (4.12–17.1 mg/g in P. lobata root, and 0.76–2.55 mg/g torigenin are those of P. thomsonii flower, which results are in P. thomsonii root) were the predominant components found consistent with those proposed by Niiho et al.’s report on the in the roots,32) both of which were not detected or detected in HPLC-ELSD analysis of nine isoflavones.18) Bebrevska et al.19) trace in the flowers. Additionally, five characteristic puerarin reported a HPLC method for the determination of three major derivatives 6″-O-xylosylpuerarin, puerarin-4′,7-O-glucoside, isoflavones, tectorigenin 7-O-xylosyl-glucoside, tectoridin and puerarin-4′-O-glucoside, puerarin-3′-methyoxy-4′-O-glucoside, tectorigenin, in the P. lobata flower, in which study, the au- and 3′-methoxypuerarin were also found in P. lobata root thentication of the used plant samples should be re-evaluated or P. thomsonii root,32) all of which were not detected in the because the tectorigenin and its derivatives are the major com- flowers. Some soy isoflavones, such as genistein, genistin and ponents of the P. thomsonii flower according to the present daidzein were detected in the roots as well,32) similarity to the study and Niiho et al.’s report. Actually, the plant materials flowers. However, some common isoflavones, such as ononin, can be easily authenticated on the basis of the significant dif- formononetin and mirificin, were found in the roots,32) but not ferences in the morphological characteristics of both flowers, in the flowers. Thus, our data is helpful for better understand- as described in herbal materials of Experiment section.28) ing of the difference of isoflavone composition between the Some soy and red clover isoflavones were also detected Pueraria roots and flowers. in both Pueraria flowers. P. lobata flower contained more Twelve saponins were simultaneously quantified in the biochanin A (0.94–1.42 mg/g), but less glycitin (not detected), study for the first time. Among them, kaikasaponin III and genistin (trace—0.25 mg/g) and genistein (trace) than P. thom- soyasaponin I were both major saponins found in both P. sonii flower (trace—0.05 mg/g for biochanin A, 0.21–6.24 mg/g lobata flower and P. thomsonii flower (1.26–15.2 mg/g for 950 Vol. 61, No. 9 kaikasaponin III, and 2.65–19.1 mg/g soyasaponin I). Kai- The VIP (variable importance in the projection) value ensured kasaponin II and kakkasaponin I were in richer abundance in the significance of potential markers. Four ions, a (tR 7.10 min, P. lobata flower (1.63–5.74 mg/g, and 5.08–12.3 mg/g) than in m/z 283.0604, VIP 20.5), b (tR 4.19 min, m/z 607.1666, VIP P. thomsonii flower (trace—2.96 mg/g, and 1.43–5.47 mg/g), 19.7), c (tR 4.72 min, m/z 475.1245, VIP 14.1), d (tR 8.16 min, suggesting that both are the characteristic saponins for P. m/z 895.5042, VIP 13.0) at the bottom left corner and two lobata flower. On the other hand, soyasaponin IV and bap- ions, e (tR 2.60 min, m/z 461.1079, VIP 29.8) and f (tR 2.35 min, tisiasaponin I were found in relatively higher amount in P. m/z 593.1492, VIP 21.9) at the top right corner of “S” are thomsonii flower (0.09–1.31 mg/g and 1.01–3.51 mg/g) than the marker compounds of P. lobata and P. thomsonii which that in P. lobata flower (0.00–0.08 mg/g and 0.09–0.86 mg/g), contribute most to difference between two Pueraria flowers, suggesting both are the characteristic saponins for P. thom- respectively. sonii flower. The rest six saponins, phaseoside IV, astragalo- The top six leading markers mentioned above were struc- side VIII, kaikasaponin I, azukisaponin I, kakkasaponin II, turally identified as biochanin A (a), kakkalide (b), irisoli- and kakkasaponin III were detected in low content in both done-O-hexoside (c), kakkasaponin I (d), tectoridin (e) and Pueraria flowers. Total contents of 12 saponins found in P. tectorigenin-7-O-xylosylglucoside (f), respectively. The results labata flower was 23.2–60.6 mg/g, and that in P. thomsonii of multivariate statistical analysis supported the quantification flower was 9.07–42.4 mg/g. Otherwise, two flavones, rutin results in our study. and luteolin, were found in small amount or trace in P. lobata flower (trace—0.09 mg/g for rutin and 0.04–0.07 mg/g for lu- Conclusion teolin) and P. thomsonii flower (0.13–4.64 mg/g for rutin and The present study is the first report on a UPLC-QTOF/MS 0.05–0.42 mg/g for luteolin). method for the rapid structural elucidation of 25 isoflavones, Several studies on HPLC analysis of the Pueraria flower 13 saponions and 3 flavones from the flowers of P. lobata and have been reported, and most of them focused on isofla- P. thomsonii. Moreover, a validated method was successfully vones.18–20,33) Although a comparative chemical profiling of applied for simultaneous quantification of 29 of them with isoflavones of the flowers of P. lobata and P. thomsonii was good accuracy and precision. The results demonstrate the sim- established, saponin composition of both flowers has been ilarity and differences in isoflavones, saponions and flavones obscure. compositions between both flower herbs, which is helpful for The present study clearly demonstrated the difference the standardization and quality control of plant materials of of chemical composition between both Pueraria flowers by Flos Puerariae. Furthermore, with multivariate statistical anal- UPLC-QTOF/MS analysis. Moreover, the established UPLC- ysis, the determined markers should be more representative. QTOF/MS profiles working in less than 10 min may be more practical for the chemical characterization of the P. lobata and Acknowledgments This study was financially supported the P. thomsonii flowers than the HPLC methods. However, a by the Research Fund for the Doctoral Program of Higher reliable fingerprint of plant herbs, and multivariate statistical Education, China (No. 200801630007), and the 2008 Scien- analysis should be established on the basis of analysis of large tific Technology Plan Project from Science and Technology amount of samples, which is a limitation in the present study, Department of Liaoning Province, China (No. 20082260233). and needs to be studied in the near future. Multivariate Statistical Analysis To further visualize Supplementary Information the difference between the UPLC-QTOF/MS profiles obtained Supplemental Data S1. Calibrations and Detection Limits from P. lobata and P. thomsonii extracts, unsupervised prin- for Marker Compounds. cipal component analysis (PCA) and supervised orthogonal Supplemental Data S2. Intra-day, Inter-day Precision and partial least squared discriminant analysis (OPLS-DA) were Recovery of 8 Major Markers. performed to process data and figure out the most important Supplemental Data S3. The details of spectroscopic data in component for their difference. Table 2 of manuscript. The score plot obtained by all observations using 8437 Pareto-scaled variables from the two species is displayed in References Fig. 5A. A clear separation can be seen between P. lobata and 1) Chinese Pharmacopeia Commission, “Pharmacopeia of the People’s P. thomsonii. 66.51% of the variables can be explained by two Republic of China,” Vol. 1, China Medical Science Publisher, Bei- indices, which were calculated by cross validation. 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