Qualitative and quantitative analyses of three bioactive compounds in different parts of Forsythia suspensa by high-performance liquid chromatography-electrospray ionization-mass spectrometry

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Microchemical Journal 89 (2008) 159 – 164 www.elsevier.com/locate/microc

Qualitative and quantitative analyses of three bioactive compounds in different parts of Forsythia suspensa by high-performance liquid chromatography-electrospray ionization-mass spectrometry Huanhuan Qu a , Baixue Li b , Xu Li c , Guangzhong Tu c , Juan Lü c , Wenji Sun a,⁎ a

Biomedicine Key Laboratory of Shaanxi Province, Northwest University, Xi'an 710069, PR China b Chengdu University of Traditional Chinese Medicine, Chengdu 610075, PR China c Beijing Institute of Microchemistry, Beijing 100091, PR China Received 5 January 2008; received in revised form 9 February 2008; accepted 9 February 2008 Available online 16 Febraury 2008

Abstract A high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analytical method was developed to simultaneously detect and quantify three main distinctive compounds (forsythiaside, rutin and forsythin) in different parts of Forsythia suspensa (F. suspensa), an herbal medicine. This was the first report on the quantification of bioactive constituents in different parts of F. suspensa by HPLC-ESI-MS analytical method. The calibration curves of the three compounds showed good linearity (R2 N 0.9994). The method was reproducible with intra- and inter-day variation less than 1.35% and 2.00%, respectively. The recovery of the assay was in the range of 98.27– 101.07%. The results indicated that the developed assay could be considered as a suitable quality control method for this commonly used herbal medicine. © 2008 Elsevier B.V. All rights reserved. Keywords: Forsythia suspensa; High-performance liquid chromatography-electrospray ionization-mass spectrometry; Forsythiaside; Rutin; Forsythin

1. Introduction Forsythia suspensa (Thunb.) Vahl. is widely distributed in China, Korea, Japan and many European nations. The fruit of this plant is a well-known traditional Chinese medicine (TCM), named “Lianqiao” in Chinese. According to maturity level of the fruits, the commercial herbs could be classified into “Qingqiao” and “Laoqiao”, and the seeds of Qingqiao are customarily called “Qiaoxin”, all of them are official sources of this TCM. More than 40 Chinese medicinal preparations containing F. suspansa are listed in Chinese Pharmacopoeia,

⁎ Corresponding author. Biomedicine Key Laboratory of Shaanxi Province, Northwest University, Xi'an 710069, PR China. Tel.: +86 29 88304569; fax: +86 29 88304368. E-mail addresses: [email protected] (H. Qu), [email protected] (W. Sun). 0026-265X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2008.02.002

such as Shuanghuanglian oral solution, Yinqiao Jiedu Tablet and Qinlian Tablet, etc. [1]. In addition, the immature leaves and flowers of this plant are used as a kind of health tea in the Orient. The crude herb had been widely used as an antipyretic, antidotal and anti-inflammatory agent for the treatment of infections, such as acute nephritis, erysipelas and ulcer [2,3]. It was also reported that F. suspensa could suppress vomiting, resist hepatic injury, inhibit elastase activity, and exhibit diuretic, analgesic, antioxidant, antiendotoxin and antiviral effects [4–8]. TCMs have attracted worldwide attention in many fields recent years, owing to their low toxicity and effective therapeutical performance with minimum side effects in many diseases [9,10]. But, the quantity and quality of efficacy data on herbal medicines are far from sufficiency to satisfy the criteria needed to support their use worldwide [11–13]. The main reason lies in the lack of the reliable and acceptable methods for the quality evaluation of TCMs.

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At present, the quality control of Lianqiao is mainly conducted according to China Pharmacopoeia (vol. 1, Edition 2005), in which only forsythin is determined by highperformance liquid chromatography-ultraviolet detector (HPLC-UV) method [1]. Interestingly, many studies [14–17] have found that the content of forsythin in leaves was much higher than in the fruits of this herb. That clearly questioned us to think whether the leaves could replace fruits as this TCM. Lately, the bioactive researches of this herb proved that forsythiaside, forsythin and rutin, among a number of compounds isolated from this plant [18], were mainly responsible for the various biological activities. For example, forsythiaside showed strong antibacterial, antiviral, antioxidant, antiinflammatory and cyclic adenosine monophosphate phosphodiesterase (cAMP) inhibitory effect [3,19–21]; forsythin showed antioxidant and weight losing abilities [22]; and rutin showed strong antioxidant effects [23]. Thus, the use of forsythin as unique marker component of quality control for F. suspensa is not enough, and sometimes leads to a biased assessment. Quantification of forsythiaside, forsythin and rutin (see Fig. 1 for their structures) in F. suspensa would be of great significance for the evaluation of the quality of this herb. Compared with HPLC-UV analytical method, HPLC-ESIMS analytical method is a more powerful approach to rapidly identify and elucidate multi-ingredients in TCMs due to its low detection limit, with high specificity and excellent ability of structure elucidation. Recently, HPLC-ESI-MS analyses have been increasingly applied for pharmaceutical research and for quality control of TCMs [24,25]. However, the HPLC-ESI-MS analyses to identify and determine bioactive compounds in different parts of F. suspensa have not been investigated yet. The current study aimed at developing a simple and feasible method for the simultaneous quantification of three marker constituents in different parts of F. suspensa in order to control the quality of this important Chinese herbal medicine.

Fig. 1. Chemical structures of three investigated compounds.

identified by Professor Yazhou Wang (Northwest University, Xi'an 710069, China). Voucher specimens were deposited in the Biology and Medicine Key Laboratory of Shaanxi province, China.

2. Materials and methods 2.3. Preparation of standard solution 2.1. Reagents and chemicals Methanol for HPLC analysis was of chromatographic grade (Merck, Darmstadt, Germany). Water for HPLC analysis was purified by a Milli-Q water purification system (Millipore Corporation, MA, USA). Other reagents were of analytical grade. Reference compounds, forsythiaside, rutin and forsythin were isolated from F. suspensa in our laboratory and their structures were characterized by UV, IR, 1H NMR, 13C NMR and MS in comparison with the data in references [26]. Their purities were determined to be more than 98% by HPLC analysis. 2.2. Plant material The roots, barks, branches, leaves, flowers, fruits, seeds and fruit shells of F. suspensa were collected in Northwest University, Xi'an, China, in 2006. The plant materials were

Three references were accurately weighed, dissolved in methanol–water (60:40, v/v) solution and diluted to appropriate concentration. Stock solution of the mixture of three references, containing forsythiaside (0.99 mg mL− 1), rutin (0.15 mg mL− 1) and forsythin (0.44 mg mL− 1) was stored in the refrigerator at 4 °C. The solution was brought to room temperature and filtered through a 0.45 μm membrane filter before HPLC analysis. 2.4. Preparation of plant samples The plant materials were powdered in a grinder to get 20mesh size powders. Each powder (1 g) of different parts of F. suspensa was ultrasound extracted with methanol (30 mL) for 30 min, then, added with 20 mL double distilled water. Last, the samples were ready for the HPLC analysis after being filtered by means of a Millipore filter (0.45 μm).

H. Qu et al. / Microchemical Journal 89 (2008) 159–164 Table 1 tR, UV λmax, structural types, and MS date of the components determined tR UV λmax Structural (min) (nm) types

[M–H]− Other negative m/z ions (m/z) a

Forsythiaside 22.5 205, 333 Phenylethanol 623.4 Rutin Forsythin a

32 210, 350 Flavonoid 35.5 215, 277 Lignan

609.1 533.1

477.3 [M–H–rha]−, 460.5 [M–H–caffeoyl]− 300.9 [M–H–glc]− 371.1 [M–H–glc]−, 356.0 [371–CH3]−

rha: rhamnose, glc: glucose.

161

negative ion mode (ESI−). Conditions of MS analysis were as follows: drying gas (N2) flow rate, 12 L min− 1; drying gas temperature, 300 °C; nebulizing gas (N2) pressure, 40 psi; capillary voltage, 3500 V; full scan range, 50–1000 m/z; fragment, 130 V. The ions were monitored in Selected IonMonitoring (SIM) mode by selecting for the ions of each target constituent shown in Table 1. Taking into account the elution times, the following starting points were selected for SIM experiments: t = 0–25 min, m/z 623; t = 25–33 min, m/z 609; t = 33–40 min, m/z 533. 3. Results and discussion

2.5. HPLC-ESI-MS analysis 3.1. Optimization of HPLC-ESI-MS system A Beckman 125 HPLC instrument (Beckman, USA), equipped with a Hamilton autosampler and a 168 photodiode array detector (DAD) was used. The UV spectra were recorded between 190 and 490 nm for peak characterization, and the detection wavelength was set at 270 nm. An Agilent ZORBAX Extend-C18 (4.6 mm × 250 mm, 5 μm) was used along with a pre-column filled with the same stationary phase. Column temperature was held at 27 °C. Elution was carried out at a flow rate of 1 mL min− 1 with mobile phases consisting of water (A) and methanol (B). The gradient elution was as follows: 0– 14 min, 34% (B), 14–15 min, 34–42% (B), 15–23 min, 42% (B), 23–24 min, 42–45% (B), 24–34 min, 45% (B), 34–36 min, 45–80% (B), and 36–40 min, 80% (B). For HPLC-MS analysis, a Bruker ESQUIRE-LC (Bruker, USA) mass spectrometer was connected to the HPLC instrument via an ESI source. The mass spectrometer was run in the

The optimization of experimental conditions was guided by the requirement of obtaining chromatograms with better resolution of adjacent peaks. Because the ingredients in the sample could not be separated with isocratic HPLC elution, gradient elution was carried out. Optimized chromatographic conditions were achieved after several trials with elution systems of acetonitrile–water, methanol–water and methanol– acetate buffer in various proportions. The optimal mobile phase, consisting of methanol–water, was subsequently employed, which led to good resolution and satisfactory peak shape. DAD detection was employed at the wavelength range of 190–490 nm. It was found that the chromatogram at 270 nm could properly represent the profile of the three target constituents, and showed good separation and high sensitivity. For the MS analytical conditions, the negative ion mode was

Fig. 2. HPLC-UV and HPLC-MS chromatograms of samples. (a) HPLC-UV chromatogram of standard solution; (b) HPLC-UV chromatogram of Qingqiao extract; (c) TIC chromatogram of standard solution; (d) TIC chromatogram of Qingqiao extract. 1, forsythiaside; 2, rutin; 3, forsythin.

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more sensitive than the positive ion mode for the three analytes.

Table 2 Statistical results of linear regression, LOD and LQD of three analytes (n = 3) Compound

3.2. Qualitative analysis Under the proposed conditions, samples were analyzed and their HPLC-UV and total ion current (TIC) profiles were given in Fig. 2. Table 1 listed retention times (tR), UV λmax, structural types, quasi-molecular ions [M–H]− and other negative fragment ions of the three target analytes. In MS spectra, [M–H]− can be easily observed, and other fragment ions of losing –rha, –glc, –caffeoyl and –CH3 were observable (as shown in Fig. 3). The fragmentation patterns were well matched with the chemical structures of corresponding references, respectively. From the retention features and m/z

Forsythiaside Rutin Forsythin a

Linear regression

LOD (µg)

LQD (µg)

2.96–49.30

0.14

0.16

0.9994

0.46–7.60

0.17

0.20

0.9998

1.32–22.00

0.08

0.15

Regression equation a

Correlation coefficient (R2)

Linear range (µg)

Y = 582121 X − 816019 Y = 2.0 × 106 X − 546202 Y = 580793 X − 33183

0.9995

Y is the peak area, X the content.

values, the three components were identified from 60% methanol extract of F. suspensa. 3.3. Method validation 3.3.1. Linearity, limits of detection (LOD) and limits of quantitation (LOQ) The linear calibration curves were constructed by six concentration assays of standard solution in triplicate. Calibration curves were constructed by plotting the integrated chromatographic peak areas (Y) versus the corresponding contents of the injected standards (X). Least square method regression was employed, and the results were presented in Table 2. High correlation coefficient values (R2 N 0.9994) were achieved in relatively wide concentration ranges for all the analytes, which indicated that this method was precise and sensitive for the quantitative evaluation. The LOD and LOQ for each analyte under the present chromatographic were calculated at signal-to-noise ratio (S/N) of 3 and 10, respectively. The results were also presented in Table 2. 3.3.2. Precision The intra-and inter-day precisions were determined by analyzing samples during a single day (n = 6) and on three consecutive days (n = 3), respectively. Variations were expressed by relative standard deviations (R.S.D.) of the peak

Table 3 Statistical results of stability and recovery Compound

Stability (n = 6)

Accuracy

R.S.D. (%)

Mean R.S.D. Added Recorded amount (mg) amount a (mg) recovery (%) (%)

Forsythiaside 1.61

Fig. 3. Mass spectra of Qingqiao extract, peak 1 (a); peak 2 (b); peak 3(c).

Rutin

1.32

Forsythin

1.96

a

8.25 12.33 24.74 1.27 1.91 3.81 3.67 5.52 11.01

8.33 ± 0.016 12.22 ± 0.077 24.93 ± 0.082 1.25 ± 0.010 1.88 ± 0.011 3.84 ± 0.008 3.71 ± 0.016 5.57 ± 0.026 10.95 ± 0.031

Mean values ± standard deviations (n = 3).

101.00 99.10 100.79 98.79 98.27 100.79 101.07 100.82 99.44

0.19 0.63 0.33 0.77 0.56 0.20 0.44 0.46 0.28

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area. The R.S.D. values of intra-day variations for forsythiaside, rutin and forsythin were 1.20, 1.35 and 1.04%; and the inter-day variations for them were 2.00, 1.63 and 0.68%, respectively. The results indicated that the method was acceptable. 3.3.3. Stability and accuracy For stability investigation, the Qingqiao sample solution stored at room temperature was determined at 0, 4, 8, 16, 24 and 48 h after extraction. The results in Table 3 showed that the R.S.D. values of peak areas were less than 1.96% (n = 6). It indicated that the extract was stable within 48 h at room temperature. To further evaluate the accuracy of the method, a recovery test was performed by spiking known quantities of the mixed standard solution to known amounts of Qingqiao samples (forsythiaside 3.579%, rutin 0.136% and forsythin 0.391%), which were determined previously. The resultant samples were then extracted and analyzed with the described method. The added standard solutions were prepared in three different concentration levels (high, medium and low) and triplicate experiments at each level. The accuracy was calculated with the value of detected versus added amounts. The recovery of the method was in the range of 98.27–101.07%, with R.S.D. values ranging from 0.19% to 0.77% (n = 3). Considering the results, the method was deemed to be accurate. 3.4. Quantitative analysis The developed HPLC-ESI-MS analytical method was applied to simultaneously determine forsythiaside, rutin and forsythin in different parts of F. suspensa. The quantitative analyses were performed by means of the external standard methods. As shown in Table 4, forsythiaside was the most abundant component in different parts of this herb and there was much more forsythiaside in barks, leaves, flowers, fruits and seeds. The content of forsythin in leaves of this herb was much higher than in the other parts. The flower was the main source of existing rutin. The variation in contents of constituents could certainly lead to the variation of therapeutic effects [27]; hence, it was not scientific to select forsythin as the only quantitative constituent for F. suspensa. Though the

Table 4 Contents of the three compounds in different parts of F. suspensa (n = 3) Parts

Roots Barks Branches Leaves Flowers Qingqiao Qiaoxin Laoqiao a b

Forsythiaside

Rutin

Content (%)

R.S.D. (%)

Content (%)

R.S.D. (%)

Content (%)

R.S.D. (%)

0.573 7.421 0.072 3.170 3.161 3.783 4.717 0.257

0.89 1.30 1.55 0.51 1.09 0.02 0.06 0.81

ND a 0.053 ND 0.667 2.125 0.105 0.100 0.167

NC b 1.60 NC 0.97 0.41 1.12 0.29 0.79

0.676 0.721 ND 2.159 0.021 0.365 0.467 0.043

0.22 1.61 NC 0.45 1.49 1.05 1.31 1.53

Not detected. Not calculated.

Forsythin

163

leaves contain much forsythin, they cannot replace fruits as this TCM. The results also indicated that the contents of the three bioactive compounds, forsythiaside, forsythin and rutin, were higher in Qingqiao than those in Laoqiao, which was in agreement with previous studies [14–17]. So the sources of this TCM used for different Chinese medicinal preparations should be strictly distinguished. 4. Conclusions The proposed method made it possible to determine the three main bioactive components, forsythiaside, forsythin and rutin, in F. suspensa in one run with acceptable levels of linearity, precision, repeatability and accuracy. This was the first report on the simultaneous quantification of the three marker constituents in different parts of F. suspensa by the HPLC-ESI-MS analytical method. The results demonstrated that the developed method could be applied as a reliable and sensitive quality control for this important Chinese herbal medicine. Acknowledgments We thank Hongwei Liu, for providing fruits of F. suspensa for the experiments and providing support in method development, and Hong Lian, for revising the text. References [1] Pharmacopoeia Commission of PRC, Pharmacopoeia of the People's Republic of China, vol. 1, Chemical Industry Press, Beijing, 2005, pp. 117–118. [2] Y. Ozaki, J. Rui, Y. Tang, M. Satake, Biol. Pharm. Bull. 20 (1997) 861–864. [3] S. Nishibe, K. Okabe, H. Tsukamoto, A. Sakushima, S. Hisada, H. Baba, T. Akisada, Chem. Pharm. Bull. 30 (1982) 4548–4553. [4] H.Y. Zhang, J. Chin. Med. Mater. 23 (2000) 657–660. [5] J.M. Prieto, M.C. Recio, R.M. Giner, S. Manez, E.M. Giner, J.L. Rios, J. Pharm. Pharmacol. 55 (2003) 1275–1282. [6] Y. Ozaki, J. Rui, Y.T. Tang, Biol. Pharm. Bull. 23 (2000) 365–367. [7] L.W. Zhang, J. Liu, P. Yang, Food Sci. 24 (2003) 122–125. [8] Q. Fu, H.L. Cui, N.J. Cui, Tianjin Med. J. 31 (2003) 161–163. [9] W.Y. Jiang, Trends Pharmacol. Sci. 26 (2005) 558–563. [10] D. Normile, Science 299 (2003) 188–190. [11] General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines, World Health Organization (WHO), Geneva, 2000. [12] FDA Guidance for Industry-Botanical Drug Products (Draft Guidance), US Food and Drug Administration, Rochville, 2000, pp. 18–20. [13] Note for Guidance on Quality of Herbal Medicinal Products, European Medicines Agency, London, 2001, pp. 6–13. [14] W.J. Li, X.E. Li, Chin. Tradit. Herb Drugs 37 (2006) 921–924. [15] Y.M. Zhao, F.R. Li, J.X. Yang, J. Liang, L.S. Zhang, Nat. Prod. Res. Dev. 17 (2005) 157–159. [16] D. Wang, Y.Y. Zhou, J. Harbin Univ. Comm. (Nat. Sci.) 20 (2004) 642–643. [17] G. Zhang, F.R. Li, F. Duan, J. Jin, Y. Guo, Z.Z. Wang, Nat. Prod. Res. Dev. 17 (2005) 790–793. [18] Q. Li, W.S. Fen, J. Henan Univ. Chin. Med. 20 (2005) 78–80. [19] S. Nishibe, K. Okabe, H. Tsukamoto, A. Sakushima, S. Hisada, Chem. Pharm. Bull. 30 (1982) 1048–1050. [20] Y. Kimura, H. Okuda, S. Nishibe, S. Arichi, Planta Med. 53 (1987) 148–153.

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