Q-TOF-MS

Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

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Qualitative and quantitative analyses of bioactive secolignans from folk medicinal plant Peperomia dindygulensis using UHPLC-UV/Q-TOF-MS Xin-zhi Wang a , Jing-yu Liang b , Hong-mei Wen a,∗ , Chen-xiao Shan a , Rui Liu a a b

College of Pharmacy, Nanjing University of Chinese Medicine, Xianlin Avenue No. 138, Nanjing 210023, China Department of Natural Medicinal Chemistry, China Pharmaceutical University, Tongjia Lane No. 24, Nanjing 210009, China

a r t i c l e

i n f o

Article history: Received 8 October 2013 Received in revised form 15 January 2014 Accepted 19 January 2014 Available online 26 January 2014 Keywords: Peperomia dindygulensis UHPLC-UV/Q-TOF-MS Secolignans Quantification Identification

a b s t r a c t Peperomia dindygulensis, with secolignans (SLs) as major bioactive constituents, is a commonly used traditional folk medicine in mainland China for treatment of stomach, liver, mammary, and esophageal cancers. However, to date, there is no method available for the qualitative and quantitative analyses of SLs in this medicinal plant. The purpose of this study was to establish a sensitive, selective, and reproducible method for rapidly profiling, identifying, and determining SLs in the whole plant of P. dindygulensis. Ultra highperformance liquid chromatography (UHPLC) coupled with ultraviolet detector (UV) and quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS) were used for this analyses. The fragmentation behaviors of different types of SLs were described. A total of thirteen SLs, including two new derivatives, were identified or tentatively characterized in P. dindygulensis samples. In addition, seven major SLs in herbal samples from different regions in China were successfully determined. The method developed in this study is suitable for the qualitative and quantitative analyses of SLs in P. dindygulensis, and may be applicable for determining or identifying SLs from other Pepermia genus plants. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Shi-chan-cao (Chinese name), the whole plant of Peperomia dindygulensis (Piperaceae), which can be found in Yunnan, Guangxi, Guizhou, and the Fujian provinces of mainland China, is a commonly used folk medicine for treatment of stomach, liver, mammary, and esophageal cancers [1]. As previously reported, the chemical compositions of this medicinal plant mainly include secolignans (SLs) [2–5], tetrahydrofuran lignans [6], polyketides [7–10], and flavonoids [11,12], among which SLs are generally considered the major active and distinctive components [3,4]. According to recent pharmacological studies, these types of compounds have been proven to exhibit significant anti-cancer [3,4,13], anti-inflammatory [14], and anti-HIV [15,16] activities. Therefore, SLs can be used as marker components in the quality control of P. dindygulensis. Currently, reports on the qualitative and quantitative analyses of SLs in P. dindygulensis are limited. To the best of our knowledge, there is no method available for the simultaneous determination of SLs to evaluate the quality of P. dindygulensis. Moreover, to

∗ Corresponding author. Tel.: +86 025 858 11839; fax: +86 025 858 11839. E-mail address: [email protected] (H.-m. Wen). 0731-7085/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2014.01.024

date, the only way to characterize and identify SLs is by nuclear magnetic resonance (NMR) spectrum. Since NMR requires a relatively large amount of purified sample for structural elucidation and the isolation of samples from plant extract is challenging and time consuming, there may be many bioactive SLs (especially those in trace concentrations) that remain uninvestigated. Thus, it is necessary to develop a rapid, sensitive, and selective method for the quantification, structural characterization, and identification of SLs from the extracts of P. dindygulensis. Currently, ultra high-performance liquid chromatography coupled with ultraviolet detector (UHPLC-UV) is routinely applied for the quantitative analyses of phytochemical constituents, since it provides much better resolution, sensitivity, and peak shape compared to HPLC analyses. In the meantime, quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS) is gradually being introduced for natural product screening and identification, since it provides both elemental compositions and fragmentation patterns in a highly sensitive and convenient way [17]. In this study, we describe a validated UHPLC-UV method for the simultaneous determination of seven major SLs in P. dindygulensis, whose chemical structures are presented in Fig. 1. This quantitative method was successfully applied to determine seven major SLs in P. dindygulensis samples from different regions of China. In addition, the relationship between structural characteristics and

2

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

R1

R2

R3

R4

-OCH3

-OH

-OCH3

-OCH3

4

-OH

-OCH3

-OCH3

-OCH3

7a

-OCH3

-OH

9a

-OCH3

-OCH3

-OCH3

-OCH3

2

!

Type A

a

11

a

13

a

-OCH2O-OCH3

-OCH2O-

-OCH2O-

-OCH2O-

R1 1

b

-OCH3

R2

R3

R4

-OH

-OH

-OCH3

-OCH3

-OCH3(-OH)

-OH(-OCH3)

-OCH3

-OCH3

a

-OCH3

-OH

-OCH2O-

10

-OCH3

-OCH3

-OCH2O-

5 6

-OCH2O-

12

-OCH2O-

Type B

3

b

8

a

R1

R2

-OCH3

-OH

-OCH2O-

Type C Fig. 1. Structures of analyzed secolignans. a Constituents with standards. b New secolignan derivatives.

MS/MS fragmentation ions of seven SL standards was investigated in detail. Based on these data, a high-resolution-and-sensitivity UHPLC/Q-TOF-MS method was established to directly elucidate the structures of SLs in the extract of P. dindygulensis. A total of thirteen SLs were identified or tentatively characterized, two of which appeared to be novel compounds. 2. Experimental 2.1. Chemicals and reagents

C

Seven (2),

SL standards, including 4 -hydroxypeperomin 2-acetoxymethyl-4 -hydroxypeperomin C (6),

4 -hydroxypeperomin B (7), peperomin F (8), peperomin C (9), peperomin B (11), and peperomin A (13) were isolated from the whole plant of P. dindygulensis in our laboratory. Their structures (Fig. 1) were unequivocally elucidated by spectroscopic methods (i.e., MS, 1 H NMR, 13 C NMR). The purity of the standards was determined to be higher than 98% by UHPLC-DAD analyses. Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Tedia (Fairfield, OH, USA). Deionized water was prepared by passing distilled water through a Milli-Q-system (Millipore, Millford, MA, USA). The silica gel used for column chromatography was purchased from Qingdao Marine Chemical Group Corporation (Qingdao, China). All other chemicals were of analytical grade.

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11 Table 1 Summary of the tested samples of P. dindygulensis.

2.5. UHPLC-UV/Q-TOF-MS conditions for qualitative and quantitative analyses

Sample

Origin

Source (date)

A

Wuyi Mountain, Fujian Province, China Putian, Fujian Province, China

Collected from the field (2012/10) Collected from the field (2012/10) Collected from the field (2012/10) Purchased from drug store in Bozhou, Anhui Province (purchased at 2012/11) Purchased from drug store in Anguo, Hebei Province (purchased at 2012/11) Collected from the field (2012/10) Collected from the field (2012/10)

B

D

Wuzhi Mountain, Hainan Province, China Yunnan Province, China

E

Guizhou Province, China

F

Xishuangbanna, Yunnan Province, China Shangsi, Guangxi Province, China

C

G

3

2.2. Plant material The whole plants of P. dindygulensis were collected from seven different regions in mainland China (Table 1). The materials were authenticated by Prof. Sheban Pu from China Pharmaceutical University. Voucher specimens were deposited at the College of Pharmacy, Nanjing University of Chinese Medicines (Nanjing, China). 2.3. Preparation of sample solution The dry plant material was pulverized and screened through 250 ␮m sieves. The obtained fine powder (1.2 g) was accurately weighed and placed in a conical flask. After adding 15 mL of acetonitrile to the flask, the mixture was sonicated for 30 min followed by centrifugation for 15 min at 5000 rpm. The supernatant was transferred to a 50 mL volumetric flask. The procedure was repeated three times and the respective supernatants were combined. The final volume was adjusted to 50 mL with acetonitrile and mixed thoroughly. Prior to injection, an adequate volume was passed through a 0.22 ␮m syringe filter. Each sample solution was injected in triplicate. 2.4. Preparation of standard solution The stock solution was prepared by accurately weighing the standard substances and dissolving them in acetonitrile. An aliquot of each stock solution was mixed and diluted with acetonitrile to make a standard mixture. This standard mixture was further diluted with acetonitrile to provide a series of six different concentrations (Table 2). All solutions were filtered through a 0.22 ␮m filter and stored at 4 ◦ C before analyses.

UHPLC-UV/Q-TOF-MS analyses was performed with a Waters ACQUITY UHPLC coupled with a diode array detector, a Q-TOF Premier, a quadrupole, and orthogonal acceleration TOF tandem MS (Waters Co., UK) that was equipped with an electrospray ionization (ESI) interface. This system was operated using MassLynx 4.1 software (Waters Co.). Chromatography was performed using a Waters ACQUITY UHPLC system equipped with a binary solvent delivery system, an autosampler, and a diode array detector. The chromatographic column used was Waters ACQUITY CSHTM C18 column (2.1 mm × 100 mm, 1.7 ␮m particle size) with a column temperature of 30 ◦ C. The mobile phase was composed of acetonitrile (A) and 0.1% formic acid (B), with the following gradient elution: 0–9.5 min, 36–41%; 9.5–11.5 min, 41–60%; 11.5–15 min 60% of A. The flow rate was kept at 0.2 mL/min and the injection volume was 2 ␮L. UV absorbance was monitored at 210 nm. Mass spectrometry was performed on a Q-TOF system (Waters Co.). The TOF data were collected between m/z 50 and 1000. The optimized conditions were desolvation gas, 600 L/h at a temperature of 250 ◦ C, cone gas, 50 L/h at a temperature of 110 ◦ C, and capillary and cone voltages of 3 kV and 35 V, respectively. The TOF mass spectrometer was calibrated routinely in the positive electrospray ionization (ESI+ ) mode using a sodium acetate solution. The MS/MS experiments were performed using variable collision energy (10–20 eV), which was optimized for each individual compound. The accurate mass and composition for the precursor and fragment ions were calculated using Masslynx 4.1 software that was incorporated in the instrument. 2.6. Method validation for quantitative analyses A series of standard solutions were prepared to determine the linearity of the compounds. The calibration curve was obtained by plotting the normal standard concentration (x) versus the peak area (y) of the analytes. The limit of detection (LOD) and the limit of quantification (LOQ) were determined by injecting a series of dilute solutions with known concentrations. LOD and LOQ were defined as the signal-to-noise ratio equal to 3 and 10, respectively. The intra-day precision of the method was evaluated by assaying the mixed standard solutions containing three different concentrations (i.e., low, medium, and high) six times over 24 h. The same procedure was performed once a day for 3 consecutive days to determine inter-day precision. Repeatability was confirmed with six independent analytical sample solutions prepared from P. dindygulensis (originated from Guizhou Province, purchased from a drug store in Anguo, sample E) and variations were expressed by relative standard deviation (RSD). The recovery experiment was carried out by adding known amounts of the seven SL standards at low (80% of the known amount),

Table 2 Calibration curves, precision, repeatability and stability test of seven analytes. Analytes

Calibration curve

R2

Linearity range (␮g/mL)

LOD (␮g/mL)

LOQ (␮g/mL)

Precision RSD (%)

Intra-day (n = 6) 2 6 7 8 9 11 13

y = 29006x − 3033.6 y = 23095x + 3812.6 y = 20239x + 3190.5 y = 20309x + 1311.5 y = 26213x − 1194.9 y = 29184x + 4555.3 y = 33742x − 787.71

0.9996 0.9998 0.9998 0.9996 0.9995 0.9998 0.9998

0.34–34.07 0.42–53.65 0.25–31.77 0.48–62.06 0.66–84.69 3.85–492.76 2.26–289.64

0.04 0.05 0.08 0.06 0.05 0.05 0.02

0.14 0.17 0.25 0.20 0.16 0.18 0.08

1.73–2.86 1.71–4.05 2.05–2.91 1.47–2.69 2.03–3.04 1.80–3.05 2.47–3.55

Repeatability RSD (%)

Stability RSD (%)

0.28 1.08 2.07 1.60 0.64 1.92 1.72

3.47 3.07 3.84 1.64 3.86 2.81 1.99

Inter-day (n = 3) 0.65–2.93 0.76–3.27 1.21–4.39 0.65–3.26 1.11–2.23 0.87–2.56 1.60–1.98

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X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

Fig. 2. UHPLC chromatograms of secolignan standards (A) and P. dindygulensis sample E (B) detected by UV at 210 nm; UHPLC-ESI/Q-TOF-MS (TIC) profiles of P. dindygulensis sample E (C).

medium (the same as the known amount), and high (120% of the known amount) levels to a certain amount of powdered herb (sample E; 0.6 g). Then, the samples were extracted and analyzed by the aforementioned method, and the experiments were repeated in triplicate at each level. The average percent of

recovery was calculated by the formula: recovery (%) = (detection amount − original amount)/addition × 100%. To investigate the stability of the sample, the sample solutions were kept in the dark at room temperature (25 ◦ C) and analyzed at different time points (0, 2, 4, 6, 8, 12, 16, and 24 h).

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

5

Table 3 Recoveries of seven analytes. Analytes

Original (␮g)

Detected (␮g)

Recovery rate (%)

RSD (%)

2

130.6

106.8 133.5 160.2

242.94 ± 2.11a 266.42 ± 1.97 282.49 ± 2.14

104.31 ± 1.98 101.74 ± 1.48 94.82 ± 1.34

1.90 1.45 1.41

6

167.24

133.44 166.8 200.16

307.55 ± 1.88 340.77 ± 3.49 356.50 ± 0.97

105.15 ± 1.41 104.04 ± 2.09 94.56 ± 0.48

1.34 2.01 0.51

7

53.67

48.82 61.02 73.22

99.80 ± 0.71 116.56 ± 0.68 123.23 ± 0.72

94.51 ± 1.46 103.07 ± 1.11 95.00 ± 0.98

1.54 1.08 1.03

8

210.24

169.68 212.1 254.52

373.02 ± 6.39 431.73 ± 3.40 461.20 ± 1.21

95.93 ± 3.77 104.43 ± 1.60 98.60 ± 0.47

3.93 1.53 0.48

9

335.24

273.6 342 410.4

622.02 ± 4.21 685.06 ± 0.98 729.06 ± 12.02

104.82 ± 1.54 102.29 ± 0.29 95.96 ± 2.93

1.47 0.28 3.05

11

3890.7

3085.8 3857.3 4628.7

7135.7 ± 8.1 7935.9 ± 60.6 8469.1 ± 191.1

105.16 ± 0.26 104.87 ± 1.57 98.91 ± 4.13

0.25 1.50 4.17

13

1096.3

870.4 1088.0 1305.6

1991.3 ± 21.7 2218.5 ± 28.0 2340.8 ± 10.2

102.83 ± 2.49 103.15 ± 2.58 95.32 ± 0.78

2.42 2.50 0.82

a

Spiked (␮g)

Mean ± S.D. (n = 3).

2.7. Extraction and isolation of two new SLs identified by UHPLC/Q-TOF-MS The dried plant material (originated from Guizhou Province, purchased from a drug store in Anguo, sample E, 2 kg) was powdered and extracted three times (2 h/each) by refluxing with 80% (v/v) EtOH (10 L/each), and then concentrated in vacuo to yield a residue. The residue was suspended in H2 O and partitioned using EtOAc. The EtOAc extract (56.8 g) was chromatographed over a silica gel, and eluted with CH2 Cl2 –MeOH (1:0–0:1) to afford five fractions (FA − FE ). FC (10.5 g) was separated by silica gel chromatography with petroleum ether–EtOAc (2:1–0:1), and five fractions (FC1 − FC5 ) were obtained. FC4 (2.5 g) was subjected to silica gel chromatography, yielding 15 subfractions (FC4-1 − FC4-15 ), and compound 1 (2.9 mg), which was purified from FC4-11 − FC4-12 by normal-phase HPLC using CH2 Cl2 –acetone (15:1) as the solvent. FC3 (1.7 g) was divided into 12 fractions (FC3-1 − FC3-12 ) by silica gel chromatography using petroleum ether–EtOAc as the solvent. Compound 3 (3.2 mg) was then obtained from FC3-11 using normal-phase HPLC [hexane–acetone (3:2)] as the solvent. NMR was used for the structural elucidation of compounds 1 and 3, and NMR spectra were recorded on Bruker ACF-300 NMR instrument (1 H: 300 MHz, 13 C: 75 MHz). Optical rotations were determined with a Perkin-Elmer 342 polarimeter in CHCl3 .

CD spectra were obtained on a JASCO J-810 CD spectrometer in MeOH, and UV spectra were recorded on a Shimadzu UV-2401 UV/VIS spectrometer in MeOH. The NMR spectra data of compounds 1 and 3 can be found in the supplementary material. 3. Results and discussion 3.1. Optimization of chromatographic and mass spectrometric conditions To obtain good separation and ideal distribution, chromatographic conditions including columns, column temperature, mobile phase, gradient program, and detection wavelength were investigated. Stationary phases of different chromatographic columns were screened, including Waters ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 ␮m particle size), ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.7 ␮m particle size), and ACQUITY CSH C18 column (2.1 mm × 100 mm, 1.7 ␮m particle size); the ACQUITY CSH C18 column showed sharper peaks and less peak tailing than the other columns. Thus, this column was selected for UHPLC analyses. Column temperatures at 25 ◦ C, 30 ◦ C, 35 ◦ C and 40 ◦ C were examined. Baseline separation of the analytes in the P. dindygulensis samples was achieved at 30 ◦ C, but the separation

Table 4 Content (␮g/g) of the seven secolignans in P. dindygulensis (n = 3). Sample/ analytes

A

B

C

D

2 6 7 8 9 11 13

82.95 ± 0.80a 82.86 ± 1.81 132.32 ± 0.66 573.13 ± 1.98 258.07 ± 4.65 3296.8 ± 41.86 2527.9 ± 22.11

26.62 ± 0.60 76.91 ± 1.24 −b +c + 2327.2 ± 47.56 +

133.08 ± 1.59 178.47 ± 4.45 98.72 ± 1.77 456.86 ± 3.93 386.33 ± 13.23 4533.9 ± 38.06 1957.2 ± 10.38

94.71 ± 0.78 127.92 ± 1.18 165.44 ± 5.72 1076.0 ± 34.80 296.73 ± 8.94 4209.5 ± 47.76 3295.8 ± 37.59

Total

5954.1

2430.8

7744.6

9266.1

a b c

Mean ± S.D. (n = 3). Not detected. Less than the quantifiable limit.

E 219.59 ± 0.49 283.27 ± 2.25 87.69 ± 1.16 356.75 ± 2.28 579.85 ± 0.94 6620.0 ± 26.86 1864.7 ± 11.49 10,012

F

G

142.29 ± 0.21 208.01 ± 3.20 105.05 ± 1.05 466.85 ± 4.13 381.53 ± 3.36 4784.5 ± 57.52 2107.0 ± 25.82

133.72 ± 2.13 179.08 ± 2.16 51.42 ± 0.63 199.29 ± 2.45 342.36 ± 0.52 3984.7 ± 20.71 1108.7 ± 4.98

8195.2

5999.3

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of some analytes was unsatisfactory at elevated temperatures up to 35 ◦ C. Accordingly, 30 ◦ C was selected as the optimal column temperature. Different mobile phases (acetonitrile–water, methanol–water), flow rates (0.2, 0.3, 0.4 mL/min), and gradient programs were tested. After various studies, a gradient mobile phase of acetonitrile–water at a flow rate of 0.2 mL/min (described in Section 2.5) was developed to achieve the best peak resolution, stable baseline, and reduced column back pressure. Formic acid (0.1%) was used to improve chromatographic behavior and facilitate MS ionization. The wavelength for UV detection was set at 210 nm because it exhibited higher sensitivity and a more stable baseline than other wavelengths. The representative UHPLC chromatograms of SL standards and P. dindygulensis samples are shown in Fig. 2. With regard to mass spectrometric conditions, both positive and negative ionization modes were investigated. The positive mode had higher sensitivity, clearer mass spectra, and lower background noise than the negative mode in confirming the precursor SL ions. Therefore, the positive ionization mode was chosen for subsequent experiments. In addition, cone voltage and collision energy were optimized because of their important roles in MS/MS fragmentation information. Considering sensitivity and maximal fragmentation information, the values of cone voltage at 35 V and collision energy in the range of 10–20 eV were applied in the MS/MS mode. 3.2. Optimization of extraction methods To obtain satisfactory extraction efficiency, key factors such as type of solvent [methanol, acetonitrile, different alcohol concentrations (70%, 80%, 95%, v/v)], extraction method (refluxing, ultrasonic, Soxhlet), extraction time (15, 30, 45, 60, 90 min), and extraction procedure (repeated two, three, or four times) were investigated. The optimized sample extraction condition was achieved by using acetonitrile in an ultrasonic water bath for 30 min; this was repeated three times. 3.3. Quantitative analyses by UHPLC-UV 3.3.1. Validation of the developed method All calibration curves showed good linearity (R2 ≥ 0.9995) and a relatively wide range of concentrations (Table 2). For the different components, the LOD ranged from 0.02 to 0.08 ␮g/mL, and the LOQ ranged from 0.08 to 0.25 ␮g/mL (Table 2). The intra-day (n = 6) and inter-day (n = 3) precision of the standard solutions were within 4.05% and 4.39% (Table 2), respectively. The sample solution remained stable for 24 h, and the RSD values were lower than 3.86%. The RSD of analyses repeatability was within 2.07% for the seven SL standards (Table 3). Recovery was calculated based on the total amount of individual target compounds. As shown in Table 3, the mean recovery rate was between 94.51% and 105.16% and the RSD values were lower than 3.93%. These results verify that the present method is selective, sensitive, accurate, precise, and reproducible for analyses of the seven SLs in P. dindygulensis samples.

Fig. 3-1. ESI-MS/MS spectra of the [M+Na]+ ion (A) and the proposed fragmentation pathway (B) of type A component peperomin B (peak 11).

quality and efficacy. Among samples collected from Yunnan (Xishuangbanna, sample F), Yunnan (Bozhou pharmaceutical store, sample D), Fujian (Wuyi Mountain, sample A), Fujian (Putian, sample B), Hainan (Wuzhi Mountain, sample C), Guizhou (Anguo pharmaceutical store, sample E), and Guangxi (Shangsi, sample G) regions in China. The total content of the seven SLs examined ranged from 2340.8 to 10,011 ␮g/g. Samples purchased from the pharmaceutical stores (sample E and D) contained the richest amounts of bioactive components, while samples collected from Fujian (Putian, sample B) had the least amount of marker compounds. The results also showed that in all plant samples, peperomin B was the highest component, with a mean content of 4295.3 ␮g/g, followed by peperomin A, with a mean content of 2350.5 ␮g/g. In summary, SLs are abundant in most P. dindygulensis herbs, and significant differences in their content can be attributed to differences in production origins. 3.4. Qualitative analyses by UHPLC/Q-TOF-MS

3.3.2. Sample analyses The verified analyses method was applied to detect the seven SLs in seven samples of P. dindygulensis collected from different production origins. The components were determined without any apparent interference from the other constituents in P. dindygulensis. Quantification of each analyte in the samples was calculated with the external standard using the calibration curves. Information regarding the content is summarized in Table 4. The results demonstrated the successful application of this UHPLC-UV assay for the quantification of seven SLs in different P. dindygulensis samples. According to Table 4, the SL content varied dramatically among different origins, which could result in differential sample

3.4.1. Fragmentation patterns of seven reference SLs All SL standards were ionized and showed strong [M+Na]+ signals in the positive ion mode. The selected precursor ions of the seven SLs were dissociated using MS/MS with different collision energies at 10–20 eV to generate a series of abundant fragment ions. To elucidate their fragmentation patterns, these SLs were classified into three types according to the substituents on their ␥butyrolactone ring. The C-2 position of type A (standards 2, 7, 9, 11, 13) was substituted with a methyl group, while type B (standard 6) was substituted with an acetoxymethyl group. In type C (standard 8), the C-1 position was substituted with a hydroxyl group. The

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

7

Table 5 MS/MS data and proposed fragmentation pathways of the seven standard secolignans. Precursor ion (m/z)

Molecular formular

Error (ppm)

2

455.1689 [M+Na]+

C23 H28 O8

1.54

7

439.1383 [M+Na]+

C22 H24 O8

−3.19

9

469.1857 [M+Na]+

C24 H30 O8

4.05

11

453.1516 [M+Na]+

C23 H26 O8

−1.99

13

437.1216 [M+Na]+

C22 H22 O8

0.92

B

6

497.1437 [M+Na]+

C24 H26 O10

C

8

497.1435 [M+Na]+

C24 H26 O10

Type A

Standards

Fragmentions (m/z)

Elem. comp.

Error (ppm)

Pathways

333.1345 279.1225 265.1056 233.1184 219.0998 181.0853 167.0702 317.1031 265.1056 263.0908 219.0998 217.0850 167.0702 165.0535 347.1523 279.1225 233.1182 181.0853 331.1172 279.1225 263.0929 233.1184 217.0850 181.0853 165.0535 315.0868 263.0908 217.0852 165.0535

C18 H21 O6 + C15 H19 O5 + C14 H17 O5 + C14 H17 O3 + C13 H15 O3 + C10 H13 O3 + C9 H11 O3 + C17 H17 O6 + C14 H17 O5 + C14 H15 O5 + C13 H15 O3 + C13 H13 O3 + C9 H11 O3 + C9 H9 O3 + C19 H23 O6 + C15 H19 O5 + C14 H17 O3 + C10 H13 O3 + C18 H19 O6 + C15 H19 O5 + C14 H15 O5 + C14 H17 O3 + C13 H13 O3 + C10 H13 O3 + C9 H9 O3 + C17 H15 O6 + C14 H15 O5 + C13 H13 O3 + C9 H9 O3 +

3.60 −0.72 −5.66 5.15 −8.22 −3.31 −0.60 3.47 −5.66 −2.28 −8.22 −4.15 −0.60 −6.66 9.79 −0.72 4.29 −3.31 −1.21 −0.72 5.70 5.15 −4.15 −3.31 −6.66 1.59 −2.28 −3.22 −6.66

[M−C5 H8 O2 ]+ [M−C8 H10 O3 ]+ [M−C9 H12 O3 ]+ [M−C8 H10 O3 –CO–H2 O]+ [M−C9 H12 O3 –CO–H2 O]+ [M−C8 H10 O3 –C5 H8 O2 ]+ [M−C9 H12 O3 –C5 H8 O2 ]+ [M−C5 H8 O2 ]+ [M−C8 H8 O3 ]+ [M−C8 H10 O3 ]+ [M−C8 H8 O3 –CO–H2 O]+ [M−C8 H10 O3 –CO–H2 O]+ [M−C8 H8 O3 –C5 H8 O2 ]+ [M−C8 H10 O3 –C5 H8 O2 ]+ [M−C5 H8 O2 ]+ [M−C9 H12 O3 ]+ [M−C9 H12 O3 –CO–H2 O]+ [M−C5 H8 O2 –C9 H12 O3 ]+ [M−C5 H8 O2 ]+ [M−C8 H8 O3 ]+ [M−C9 H12 O3 ]+ [M−C8 H8 O3 –CO–H2 O]+ [M−C9 H12 O3 –CO–H2 O]+ [M−C5 H8 O2 –C8 H8 O3 ]+ [M−C9 H12 O3 –C5 H8 O2 ]+ [M−C5 H8 O2 ]+ [M−C8 H8 O3 ]+ [M−C8 H8 O3 –CO–H2 O]+ [M−C8 H8 O3 –C5 H8 O2 ]+

2.61

415.1406 397.1037 317.1031 263.0908 261.0737 205.0844 167.0702 165.0535

C22 H23 O8 + C22 H21 O7 + C17 H17 O6 + C14 H15 O5 + C14 H13 O5 + C12 H13 O3 + C9 H11 O3 + C9 H9 O3 +

4.58 6.30 3.47 −2.28 −7.66 −7.31 −0.60 −6.66

[M−CH3 CO–H2 O+H]+ [M−CH3 CO–2H2 O]+ [M−CH3 CO–H2 O–C5 H6 O2 ]+ [M−CH3 CO–H2 O–C8 H10 O3 ]+ [M−CH3 CO–H2 O–C8 H8 O3 ]+ [M−CH3 CO–CO–2H2 O–CH2 ]+ [M−CH3 CO–H2 O–C8 H10 O3 –C5 H6 O2 ]+ [M−CH3 CO–H2 O–C8 H8 O3 –C5 H6 O2 ]+

2.21

397.1307 379.1198 367.1211 327.0881 315.0885 245.0810 215.0706 165.0535

C22 H21 O7 + C22 H19 O6 + C21 H19 O6 + C18 H15 O6 + C17 H15 O6 + C14 H13 O4 + C13 H11 O3 + C9 H9 O3 +

6.29 5.80 7.90 5.50 6.98 0.82 1.39 −6.66

[M−CH3 CO–2H2 O]+ [M−CH3 CO–3H2 O]+ [M−CH3 CO–3H2 O–CH2 +H]+ [M−CH3 CO–3H2 O–C4 H4 ]+ [M−CH3 CO–3H2 O–C5 H6 ]+ [M−CH3 CO–2H2 O–C8 H8 O3 ]+ [M−CH3 CO–3H2 O–CH2 –C8 H8 O3 ]+ [M−CH3 CO–3H2 O–CH2 –2C8 H8 O3 ]+

MS data of the seven SLs and their characteristic fragment ions in MS/MS spectra are summarized in Table 5. The fragmentation pathways of each group are depicted in detail below. Type A The MS/MS spectrum of peperomin B (11) in Fig. 3-1 was representative of the fragmentation pathways of type A SLs, and showed that the precursor ion of 11 (m/z 453 [M+Na]+ ) had fragment ions arising from three separate fragmentation pathways (Fig. 3-1). In the first pathway, elimination of a trimethoxyphenyl moiety from 11 produced the m/z 263 ion. Due to its good stability, the signal of this ion was quite intense. Furthermore, by consecutive neutral loss of CO and H2 O moieties from the ␥-butyrolactone ring, the m/z 217 ion was produced. In the second pathway, direct elimination of a methyleneoxymethoxyphenyl moiety from the precursor ion yielded the ion at m/z 279, after which successive loss of CO and H2 O moieties generated the ion at m/z 233. In the third pathway, elimination of the ␥-butyrolactone ring produced the ion at m/z 331, after which successive loss of one of the aromatic moiety generated ions at m/z 181 or 165. The ions at m/z 181 and 165 were of diagnostic value in cases where the two aromatic moieties showed different substitutions. Similar fragmentation pathways were observed for

4 -hydroxypeperomin C (2), 4 -hydroxypeperomin B (7), peperomin C (9), and peperomin A (13). Their MS/MS spectra are shown in Supplementary Fig. S1. Type B As shown in Fig. 3-2, type B component 2-acetoxymethyl-4 hydroxypeperomin C (6) showed a remarkable [M+Na]+ ion in the MS/MS spectra. The consecutive elimination of CH3 CO and H2 O moieties at C-2 of the precursor ion produced the typical fragment ion (at m/z 415) of 6. This characteristic ion was further broken down in three separate pathways. First, by elimination of one of the aromatic groups, ions at m/z 263 and 261 were produced. Successive loss of moieties like methylene, CO, and H2 O from the 2-methylene-␥-butyrolactone ring of the m/z 263 ion generated ion production at m/z 205. Second, successive loss of the 2-methylene␥-butyrolactone ring and one of the aromatic moieties generated ions at m/z 317, 167, and 165, respectively. Third, by neutral elimination of a H2 O moiety from the 2-methylene-␥-butyrolactone ring, a product ion at m/z 397 was produced. Type C Peperomin F (8) is a typical type C component. Due to the existence of a tetrahydrofuran ring, the fragmentation pathway of type

8

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

Fig. 3-2. ESI-MS/MS spectra of the [M+Na]+ ion (A) and the proposed fragmentation pathway (B) of type B component 2-acetoxymethyl-4 -hydroxypeperomin C (peak 6).

C compounds was quite different from those of the above SLs. As shown in Fig. 3-3, the precursor ion of 8 (m/z 497 [M+Na]+ ) produced an intense ion at m/z 397 by consecutive loss of CH3 CO and two H2 O groups at C-2 and C-1 from the tetrahydrofuran ring. This was followed by loss of one of the aromatic moieties, after which a product ion at m/z 245 was yielded. Meanwhile, ions at m/z 379, 367, and 215 were produced by consecutive loss of H2 O, methylene, and one of the aromatic groups from the base peak ion at m/z 397. Then, product ions at m/z 327 and 315 were observed in the MS/MS spectra by successive loss of C3 H2 and CH2 moieties from the ion at m/z 367. Finally, by further elimination of a methyleneoxymethoxyphenyl moiety from the product ion at m/z 315, an aromatic ion at m/z 165 was generated. 3.4.2. Identification of SLs from the plant samples The total ion current (TIC) chromatogram of an extract of P. dindygulensis (sample E) is shown in Fig. 2. According to the LC retention behavior (tR ), the UV and MS/MS spectrum obtained online, 13 SLs, including two new ones, were identified or tentatively characterized. The retention time (tR ), molecular weight (MW), and maximal UV wavelength of each SLs detected in the P. dindygulensis samples are presented in Table 6. The SLs displayed a strong absorption at a wavelength of 206–220 nm, and weak absorptions at 243–252 nm. 3.4.2.1. Identification of type A SLs. Peaks 2 (tR = 4.34 min), 7 (tR = 6.71 min), 9 (tR = 7.54 min), 11 (tR = 10.09 min), and 13 (tR = 11.48 min) in the extract of P. dindygulensis samples were identified as being 4 -hydroxypeperomin C, 4 -hydroxypeperomin

Fig. 3-3. ESI-MS/MS spectra of the [M+Na]+ ion (A) and the proposed fragmentation pathway (B) of type C component peperomin F (peak 8).

B, peperomin C, peperomin B, and peperomin A, respectively, by comparison of their MS/MS data, tR , and UV spectra with those of the five standards. The UV and MS/MS spectra of peak 4 (tR = 5.01 min) and peak 2 (tR = 4.34 min) were identical, but the LC retention behaviors differed, suggesting the different substituted position of a hydroxyl group at the aromatic rings (the precise position of the hydroxyl group cannot be fully elucidated by the m/z 167 ion in the MS/MS spectra). After a thorough literature search, peak 4 was preliminarily assigned as 2-methyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(3 -hydroxy4 ,5 -dimethoxyphenyl)methyl]butyrolactone, which was previously isolated and identified from P. dindygulensis by Wu et al. [3]. 3.4.2.2. Identification of type B SLs. Except for 2-acetoxymethyl4 -hydroxypeperomin C (peak 6, tR = 5.91 min), four type B SLs were credibly identified, one of which was a novel compound. Peak 1 (tR = 3.21 min) gave an [M+Na]+ ion at m/z 499. An initial loss of 60 Da produced a prominent product ion at m/z 439 [M−CH3 CO–H2 O+Na]+ , followed by loss of 18 Da to form an ion at m/z 399 [M−CH3 CO–2H2 O+Na]+ . Obvious losses of 120 Da, 162 Da, and 190 Da from the m/z 439 ion were observed to yield product ions at m/z 319 [M−CH3 CO–H2 O–C5 H6 O2 ]+ , 277 [M−CH3 CO–H2 O–C7 H8 O3 ]+ , and 249 [M−CH3 CO–H2 O–C8 H10 O3 ]+ , respectively. Then two product ions at m/z 181 and 153 were generated by the precursor ion at m/z 319. This fragmentation pattern was very similar to peak 6 (Table 6), with the exception of the substitution groups at the aromatic rings (Fig. 4A). Since the product ions at m/z 181 and 153 were the diagnostic value of the aromatic substitution, we deduced that a dihydroxymethoxyphenyl and trimethoxyphenyl moiety existed in peak 1.

Table 6 The secolignans detected by UPLC-DAD/Q-TOF-MS from the extracts of P. dindygulensis. Peak no.

tR (min)

max (nm)

Precursor ion (m/z)

Elem. Comp.

Error (ppm)

Fragment ions (m/z)

Identification

−0.80

439.1463, 399.1495, 319.1142, 277.1099, 249.0793, 203.0723, 181.0853, 153.0522 333.1375, 279.1225, 265.1056, 219.0998, 233.1184, 181.0853, 167.0702 441.1577, 399.1497, 381.1333, 369.1353, 317.1031, 229.0837, 167.0702, 165.0535 333.1286, 279.1225, 265.1135, 233.1176, 219.1033, 181.0893, 167.0725 455.1685, 413.1571, 395.1423, 383.1482, 331.1172, 245.1154, 181.0859, 165.0535 415.1436, 397.1307, 317.1031, 263.0908, 261.0737, 205.0844, 167.0702, 165.0535 317.1031, 265.1056, 263.0908, 219.0998, 217.0850, 165.0535, 167.0702 397.1337, 379.1218, 367.1211, 327.0881, 315.0885, 245.0810, 215.0706, 165.0535 347.1523, 279.1225, 233.1202, 181.0853 429.1534, 411.1454, 331.1183, 277.1119, 261.0758, 219.1033, 181.0839, 165.0535 331.1172, 279.1225, 263.0929, 233.1184, 217.0850, 181.0853, 165.0535 413.1272, 395.1130, 261.0837, 203.0723, 165.0535 315.0868, 263.0908, 217.0852, 165.0535

2-Acetoxymethyl-3-[(3 ,4 ,5 trimethoxyphenyl)(3 ,4 -dihydroxy-5 methoxyphenyl)methyl]butyrolactonea

1

3.21

B

206, 245

499.1571

C24 H28 O10 Na+

2

4.34

A

207, 243

455.1689

C23 H28 O8 Na+

3

4.70

C

207, 245

499.1571

C24 H28 O10 Na+

4

5.01

A

211, 244

455.1689

C23 H28 O8 Na+

1.54

5

5.51

B

219, 250

513.1721

C25 H30 O10 Na+

1.95

6

5.91

B

220, 249

497.1437

C24 H26 O10 Na+

3.82

7

6.71

A

219, 248

439.1362

C22 H24 O8 Na+

8

7.11

C

212, 252

497.1437

C24 H26 O10 Na+

3.82

9

7.54

A

207, 243

469.1857

C24 H30 O8 Na+

4.04

10

9.62

B

219, 250

511.1606

C25 H28 O10 Na+

6.06

11

10.09

A

209, 249

453.1516

C23 H26 O8 Na+

12

11.55

B

212, 252

495.1265

C24 H24 O10 Na+

0.61

13

11.82

A

219, 250

437.1214

C22 H22 O8 Na+

0.46

a b

1.54

−0.80

−1.59

−1.98

4 -Hydroxypeperomin Cb

2-Acetoxymethyl-3-[(3 ,4 ,5 trimethoxyphenyl)(4 -hydroxy-3 ,5 dimethoxyphenyl)methyl]tetrahydrofuran1-ola 2-Methyl-3-[(3 ,4 ,5 trimethoxyphenyl)(3 -hydroxy-4 ,5 dimethoxyphenyl)methyl]butyrolactone

Ref.

[3]

[3]

2-Acetoxymethyl-3{(3 ,4 ,5 trimethoxyphenyl)[3 (4 )hydroxy-4 (3 ),5 dimethoxyphenyl]methyl}butyrolactone 2-Acetoxymethyl-4 -hydroxypeperomin Cb

[3,4]

4 -Hydroxypeperomin Bb

[3]

Peperomin Fb

[2,3]

Peperomin Cb

[3,5,18]

2-Acetoxymethyl-3-[(3 ,4 ,5 trimethoxyphenyl)(5 -methoxy-3 ,4 methyleneoxyphenyl)methyl]butyrolactone

[3]

Peperomin Bb

[3,5,18]

2-Acetoxymethyl-3-[bis(5-methoxy-3,4methyleneoxyphenyl)methyl]butyrolactone

[3]

Peperomin Ab

[3,5,18]

[3]

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

Type

New secolignan derivatives. Positively identified via comparison with standard secolignans.

9

10

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

Fig. 4. ESI-MS/MS spectrum of two new secolignan derivatives peak 1 (A) and 3 (B).

Therefore, peak 1 was tentatively identified as 2-acetoxymethyl-3[(3 ,4 ,5 -trimethoxyphenyl) (3 ,4 -dihydroxy-5 -methoxyphenyl) or 2-acetoxymethyl-3methyl]butyrolactone [(3 ,4 ,5 -trimethoxyphenyl)(3 ,5 -dihydroxy-4 methoxyphenyl)methyl]butyrolactone by MS/MS spectra, neither of which have been previously reported. Further NMR analyses are needed to validate the chemical structure of this new compound. The fragmentation patterns of peak 5 (tR = 5.51 min), 10 (tR = 9.62 min) and 12 (tR = 11.55 min) were also similar to those of peak 6, and their MS/MS spectra are presented in Supplementary Fig. S2. A set of distinct product ions at m/z 181 and 167 were observed in the MS/MS spectra, suggesting the presence of a dimethoxyhydroxyphenyl and trimethoxyphenyl moiety in peak 5. However, the position of a hydroxyl group on the dimethoxyhydroxyphenyl moiety could not be fully elucidated by MS/MS, so peak 5 was tentatively identified as 2-Acetoxymethyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(3 hydroxy-4 ,5 -dimethoxyphenyl)methyl]butyrolactone 2-Acetoxymethyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(4 or hydroxy-3 ,5 -dimethoxyphenyl)methyl]butyrolactone, both of which have been reported [3,4] in the literature. Similarly, the compounds at peaks 10 and 12 were identified as 2-Acetoxymethyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(5

-methoxy-3 ,4 -methyleneoxyphenyl)methyl]butyrolactone and 2-Acetoxymethyl-3-[bis(5-methoxy-3,4-methyleneoxyphenyl) methyl]butyrolactone, respectively, by analyzing the diagnostic product ions at m/z 181, 165 (peak 10) and 165 (peak 12) from their MS/MS spectra. 3.4.2.3. Identification of type C SLs. In addition to peperomin F (peak 8, tR = 7.11 min), only one type C compound (peak 3, tR = 4.70 min) was detected. The precursor ion (m/z 499 [M+Na]+ ) of peak 3 yielded prominent product ions at m/z 399 [M−CH3 CO–2H2 O]+ and 381 [M−CH3 CO–3H2 O]+ , respectively. Then, product ions at m/z 369 [M−CH3 CO–3H2 O–CH2 +H]+ , 317 [M−CH3 CO–3H2 O–C5 H6 ]+ and 229 [M−CH3 CO–3H2 O–CH2 –C8 H10 O3 ]+ were generated by the precursor ion at m/z 381. This was consistent with the fragmentation characteristic of peperomin F (Fig. 3-3). The product ions at m/z 167 and 165 were observed in the MS/MS spectrum of peak 3, suggesting the possible presence of a dimethoxyhydroxyphenyl and methyleneoxymethoxy phenyl moiety. Therefore, peak 3 was tentatively identified as 2-Acetoxymethyl-3-[(5 -methoxy-3 ,4 -methyleneoxyphenyl)(4 hydroxy-3 ,5 -dimethoxyphenyl)methyl]tetrahydrofuran-1-ol or 2-Acetoxymethyl-3-[(5 -methoxy-3 ,4 -methyleneoxyphenyl)(3 hydroxy-4 ,5 -dimethoxyphenyl)methyl]tetrahydrofuran-1-ol by

X.-z. Wang et al. / Journal of Pharmaceutical and Biomedical Analysis 94 (2014) 1–11

MS/MS spectra, neither of which have been previously reported. To confirm the position of the hydroxyl group on the dimethoxyhydroxyphenyl moiety of this new component, further NMR experiments are required. 3.5. Structural validation of two new SLs by NMR spectra Peak 1 had NMR signals (supplementary material) similar to of 2-Acetoxymethyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(3 those   hydroxy-4 ,5 -dimethoxyphenyl)methyl]butyrolactone, as reported by Wu et al. [3], with the exception of the 3-hydroxy-4,5dimethoxyphenyl group. One methoxy group disappeared and MS fragments at m/z 153 suggested that a dihydroxymethoxyphenyl group existed. Instead of signals representing symmetrical aromatic protons, two nonequivalent aromatic protons [ıH 6.42 (1H, d, J = 1.5 Hz, H-2 ), 6.47 (1H, d, J = 1.5 Hz, H-6 )] were observed in the 1 H NMR of 1, so a 3,4-dihydroxy-5-dimethoxyphenyl group existed in 1. Thus, peak 1 was established as 2Acetoxymethyl-3-[(3 ,4 -dihydroxy-5 -methoxyphenyl)(3 ,4 ,5 trimethoxyphenyl)methyl]butyrolactone. The NOE cross-peak between H-2 and H-3 indicated their cis-configuration. It was a levorotatory isomer. The 1 H NMR and 13 C NMR spectra of peak 3 (supplementary material) were similar to those of peperomin F reported by Govindachari et al. [2], with the exception of the 5-methoxy3,4-methylendioxyphenyl group. One methylenedioxy group disappeared, one additional methoxy group appeared, and MS fragment at m/z 167 indicated the existence of a hydroxydimethoxyphenyl group. Two symmetrical aromatic protons [ıH 6.45 (2H, s, H-2 , 6 )] and two symmetrical methoxy groups [ıH 3.87 (6H, s, 3 , 5 -OCH3 )] were observed in the 1 H NMR, suggesting a 4hydroxy-3, 5-dimethoxyphenyl group in 3. Thus, peak 3 was finally identified as 2-Acetoxymethyl-3-[(3 ,4 ,5 -trimethoxyphenyl)(4 hydroxy-3 ,5 -dimethoxyphenyl)methyl]tetrahydro furan-1-ol. The NOE cross-peak between H-2 and H-3 indicated their cisconfiguration. The singlet of H-1 suggested that the dihedral angle H1–C1–C2–H2 was nearly 90◦ , which indicated a trans-orientation of H-1 and H-2. It was a levorotatory isomer. 4. Conclusions In this study, a UHPLC-UV method was developed for the simultaneous determination of seven bioactive SLs in P. dindygulensis for the first time. The results showed that the developed method had good linearity, precision, accuracy, and sensitivity for determination of the seven SLs. This validated method was successfully applied to simultaneously determine the seven SLs in P. dindygulensis samples from different areas of China, which laid a solid foundation for the establishment of a comprehensive quality control method for P. dindygulensis. At the same time, an UHPLC/Q-TOF-MS method for the rapid identification and structural characterization of SLs in P. dindygulensis samples was also developed. The fragmentation patterns of seven SL standards (divided into three types) were investigated for the first time in this work. The considerable fragmentation information obtained by MS/MS was very useful for structure elucidation. According to

11

the results in Table 6, thirteen SLs, including two new derivatives, were screened from the P. dindygulensis samples. This qualitative identification and structural elucidation method provide essential data for further chemical or pharmacological studies of P. dindygulenesis, and may be applied for the identification of bioactive SLs from other Pepermia genus plants. Acknowledgments This work was supported by Grants from the Natural Science Foundation of Jiangsu Province (No. BK20130954) and Natural Science Foundation of Nanjing University of Chinese Medicines for young scholars (No. 13XZR15). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.01.024. References [1] Nanjing New Medical School, Dictionary of Chinese Herbal Drugs, Shanghai Science and Technology Press, Shanghai, 1978, pp. 622. [2] T.R. Govindachari, G.N. Krishna Kumari, P.D. Partho, Two secolignans from Peperomia dindygulensis, Phytochemistry 49 (1998) 2129–2131. [3] J.L. Wu, N. Li, T. Hasegawa, J. Sakai, T. Mitsui, H. Ogura, T. Kataoka, S. Oka, M. Kiuchi, A. Tomida, T. Turuo, M. Li, W. Tang, M. Ando, Bioactive secolignans from Peperomia dindygulensis, J. Nat. Prod. 69 (2006) 790–794. [4] M.G. Lin, D.H. Yu, Q.W. Wang, Q. Lu, W.J. Zhu, F. Bai, G.X. Li, X.W. Wang, Y.F. Yang, X.M. Qin, C. Fang, H.Z. Chen, G.H. Yang, Secolignans with antiangiogenic activities from Peperomia dindygulensis, Chem. Biodivers. 8 (2011) 862–871. [5] L. Chen, Y. Yu, J.X. Dong, Chemical constituents of Peperomia dindygulensis, Chin. Tradit. Herbal Drugs 38 (2007) 491–493. [6] J.L. Wu, N. Li, T. Hasegawa, J. Sakai, S. Kakuta, W. Tang, S. Oka, M. Kiuchi, H. Ogura, T. Kataoka, A. Tomida, T. Tsuruo, M. Ando, Bioactive tetrahydrofuran lignans from Peperomia dindygulensis, J. Nat. Prod. 68 (2005) 1656–1660. [7] Q.W. Wang, D.H. Du, M.G. Lin, M. Zhao, W.J. Zhu, Q. Lu, Antiangiogenic polyketides from Peperomia dindygulensis Miq., Molecules 17 (2012) 4474–4483. [8] X.Z. Wang, W. Qu, J.Y. Liang, New long-chain aliphatic compounds from Peperomia dindygulensis, Nat. Prod. Res. 27 (2013) 796–803. [9] X.Z. Wang, W. Qu, J.Y. Liang, Two N-containing polyketide derivatives from Peperomia dindygulensis, Chem. Nat. Compd. 48 (2012) 1027–1030. [10] W.J. Zhu, M.G. Lin, G.H. Yang, Q.W. Wang, Y.F. Yang, Two novel polyketides from Peperomia dindygulensis, Chin. Tradit. Herbal Drugs 38 (2011) 420–423. [11] L. Chen, Y. Zhou, J.X. Dong, Three new flavonoid glycosides from Peperomia dindygulensis, Acta Pharm. Sin. 42 (2007) 183–186. [12] L. Chen, Y. Zhou, Y.L. Zhou, J.X. Dong, A new C-glycosylflavone from Peperomia dindygulensis, Chin. J. Chin. Mater. Med. 33 (2008) 772–774. [13] S. Xu, N. Li, M.M. Ning, C.H. Zhou, Q.R. Yang, M.W. Wang, Bioactive compounds from Peperomia pellucida, J. Nat. Prod. 69 (2006) 247–250. [14] C. Tsutsui, Y. Yamada, M. Ando, D. Toyama, J.L. Wu, L. Wang, S. Taketani, T. Kataoka, Peperomins as anti-inflammatory agents that inhibit the NF-kappaB signaling pathway, Bioorg. Med. Chem. Lett. 19 (2009) 4084–4087. [15] X.J. Zhang, G.Y. Yang, R.R. Wang, J.X. Pu, H.D. Sun, W.L. Xiao, Y.T. Zheng, 7,8Secolignans from Schisandra wilsoniana and their anti-HIV-1 activities, Chem. Biodivers. 7 (2010) 2962–27010. [16] Q.F. Hu, H.X. Mu, H.T. Huang, H.Y. Lv, S.L. Li, P.F. Tu, G.P. Li, Secolignans, neolignans and phenylpropanoids from Daphne feddei and their biological activities, Chem. Pharm. Bull. 59 (2011) 1421–1424. [17] H. Wu, J. Guo, S. Chen, X. Liu, Y. Zhou, X. Zhang, X. Xu, Recent developments in qualitative and quantitative analysis of phytochemical constituents and their metabolites using liquid chromatography–ass spectrometry, J. Pharm. Biomed. Anal. 72 (2013) 267–291. [18] C.M. Chen, F.Y. Jan, M.T. Chen, T.J. Lee, Peperomins A, B, and C, novel secolignans from Peperomia japonica, Hetrocycles 29 (1989) 411–414.