High performance liquid chromatography time of flight electrospray ionization mass spectrometry for quantification of sesquiterpenes in Chrysanthemi indici Flos active extract.

BACKGROUND: Chrysanthemi indici Flos, a traditional herbal medicine is used to clearing heat-toxicity, removing the liver fire, and improving eyesight. In our preliminary work, an active extract of CTC in C. An indici Flos with anti-hepatitis B virus and liver protective activity was found by HepG2.2.1.5 test and experiment of protein synthesis in mice's injured liver. In this work, we aimed to study the active faction CTC further by qualitative and quantitative analysis method.
MATERIALS AND METHODS: High performance liquid chromatography time of flight electrospray ionization mass spectrometry (HPLC TOF ESI-MS) analysis method of the CTC was established. Cumambrin A and angeloylcumambrin B in CTC were analyzed by high performance liquid chromatography-ultraviolet-evaporative light scattering detector (HPLC-UV-ELSD) analysis methods. A binary gradient elution program was conducted for chromatographic separation with acetonitrile (A) and ultrapure water (B) as follows: 0-10 min, 42-46% A; 10-20 min, 46-55% A; 20-25 min, 55-60% A; and 25-35 min, 60-75% A. The column temperature and UV wavelength were set at 30°C and 205 nm. RESULT: Ten constituents including (3R, 5R, 6S, 7S, 10R)-7-(2-hydroxy-2-propyl)-10-methyl-4-methyleneperhy, dronaphthal-ene-3, 5, 6-triol acetone solvate; (+)-edusmance-4, (14)-ene-11, 13-diol; linarin; luteolin; apigenin; tricin; 5, 3',4'- trimethyl-6,7-dimethoxy flavones; cumambrin A; acacetin; and angeloylcumambrin B in CTC were identified by HPLC TOF ESI-MS. The contents of sesquiterpenes in CTC were decreased by storing years.
CONCLUSIONS: The results showed that both UV and ELSD methods were feasible, accurate, and the determination results were in good consistency. The contents of two sesquiterpenes decreased with storing years. Two sesquiterpenes could be used as quality control for C. indici flos CTC.

INTRODUCTION

Chrysanthemi indici Flos (Chrysanthemum indicum L.), a traditional herbal medicine for clearing heat–toxicity, removing the liver fire and improving eyesight have a long application history, and wide distribution in China.[1] C. indici Flos is also well-known and usually used as Chrysanthemum tea, Chrysanthemum pillow, food additives, medicated bath in the folk.[234]

In our preliminary work, an active extract of (CTC) in C. indici Flos with anti-hepatitis B virus and liver protective activity was found by HepG2.2.1.5 test and experiment of protein synthesis in mice's injured liver. The results of HepG2.2.1.5 test show that the inhibition ratio to expression of hepatitis B surface antigen was 92.2% (40 μg/mL), and the inhibition ratio to expression of hepatitis B e antigen was 94.2% (40 μg/mL).[5] Then, the influence of CTC on liver protein synthesis action injured by D-amino galactose was analyzed by the levels of total protein (TP), albumin (ALB), and alanine transaminase (ALT) in serum, liver, and spleen indexes.[6] It made us excited that the different dose groups of CTC could reduc e the liver, spleen indexes, and ALT level but enhance TP and ALB levels, especially the high dose group of CTC [Figure 1]. Given the promising bioactivities of C. indici extract CTC investigations on the chemical constituents and quality control methods were very meaningful. Systematical analysis on the constituents of the active extract indicated that CTC mainly contained sesquiterpenes (about 41.7%) and flavonoids (38.8%).[7]

Figure 1

Bifendate Pills group, active extract in Chrysanthemi indici Flos low dosage group, active extract in Chrysanthemi indici Flos middle dosage group, active extract in Chrysanthemi indici Flos high dosage group. Left: Effects of active extract in Chrysanthemi indici Flos on the index of liver and spleen in mice injured by D-gal. Right: Effects of active extract in Chrysanthemi indici Flos on the total protein, albumin, and alanine transaminase in serum of mice injured by D-gal

Up till now, there are some reports on analysis methods of the active constituents in C. indici Flos. However, most of them mainly focused on the contents of flavonoids and volatile oils such as luteolin, linarin, camphor, and borneol.[891011] There is no paper reported on the contents of active sesquiterpenes[1213141516] in C. indici Flos. As we have studies, the content of flavonoids such as luteolin and linarin. Hence, in this paper, the high performance liquid chromatography-ultraviolet-evaporative light scattering detector (HPLC-UV-ELSD) analysis methods were established to determinate the contents of two significant active sesquiterpenes, cumambrin A, and angeloylcumambrin B in CTC. The results showed that the developed methods were convenient and accurate.

MATERIALS AND METHODS

Plant Material

C. indici Flos were obtained from Xinyang Henan province and identified by professor Yuefeng Bi. Voucher specimens (No. 201110) were deposited at the herbarium in Zhengzhou University School of Pharmaceutical Science (Zhengzhou, China). The dried C. indici Flos were powdered and passed through a 40-mesh sieve before extraction.

A standard procedure for active extract in C. indici Flos preparation

Air-dried flowers (3.9 kg) were extracted with 70% EtOH (3 × 6 l) by a tissue-smashing extractor, and the combined extracts were concentrated (yield 1.07 kg). The residue was suspended in H2O (5 l) and then partitioned successively with petroleum ether, petroleum ether (60–90°0)/AcOEt 1:1, and AcOEt (5 × 4 l). The petroleum ether/AcOEt 1:1 fraction was separately concentrated which afforded 46.6 g of extract. This extract is CTC. The CTC (42.1 mg) was dissolved in methanol (25 mL) and filtered through a nylon membrane filter (0.22 μm micro-pore, phenomenex, USA) for HPLC analysis.

Chemical reagents

HPLC grade acetonitrile (MeCN) and MeOH were purchased from Merck (Darmstadt, Germany). Ultrapure water was prepared by using a Millipore Milli-Q-Plus system (Bedford, MA, USA). (3R, 5R, 6S, 7S, 10R)-7-(2-hydroxy-2-propyl)-10-methyl-4-methyleneperhy, dronaphthal-ene-3, 5, 6-triol acetone solvate; (+)-edusmance-4, (14)-ene-11, 13-diol; linarin; luteolin; apigenin; tricin; 5, 3’,4’-trimethyl-6, 7-dimethoxy flavones; cumambrin A; acacetin; angeloylcumambrin B were prepared and purified by our lab. The purity was > 98%. The chemical structures of all reference substances are shown in Figure 2.

Figure 2

The high performance liquid chromatography chromatograms of active extract in Chrysanthemi indici Flos and structures formulas of Peak 1, (3R, 5R, 6S, 7S, 10R)-7-(2-hydroxy-2-propyl)-10-methyl-4-methyleneperhy, dronaphthal-ene-3, 5, 6-triol acetone solvate; Peak 2, (+)-edusmance-4, (14)-ene-11, 13-diol; Peak 3, linarin; Peak 4: Luteolin; Peak 5, apigenin; Peak 6, tricin; Peak 7, 5, 3’, 4’-trimethyl-6,7-dimethoxy flavones; Peak 8, cumambrin A; Peak 9, acacetin; Peak 10, angeloylcumambrin B

Standard solutions

A mixed standard stock solution containing 10 reference substances was prepared in MeOH for HPLC analysis. Another mixed stock solution of cumambrin A and angeloylcumambrin B reference substances was prepared in MeOH and further diluted to six data points, and each standard dilution was injected in triplicate for the calibration curve. All solutions were passed through a 0.22 μm micropore membrane before injection and stored at 4°C.

Qualitative analysis of the active extract in Chrysanthemi indici Flos by high performance liquid chromatography time of flight electrospray ionization mass spectrometry

High performance liquid chromatography time of flight electrospray ionization mass spectrometry (HPLC TOF ESI-MS) analysis of CTC was performed on Waters Xevo G2 Q-TOF (Milford, MA, USA). A YMC column (250 mm *4.6 mm i.d., 5 μm particles) was used to separate the CTC. A gradient elution was used for chromatographic separation with 0.5% methanoic acid (A, pH 3, 0.1 M) and MeCN (B) as follow: 0–30 min, 25–35% B; 30–35 min, 35–43% B; 35–50 min, 43–45% B; 50–65 min, 45–57% B; and 65–80 min, 57–70% B. The flow rate and the injection volume were 0.8 mL/min and 10 μL, respectively. The column temperature and wavelength were set at 35°C and 205 nm. For MS detection, a positive ion mode ESI was used. Nitrogen was used as the desolation and cone gas with the flow rate of 50 L/h. The optimal MS parameters were as follows: Capillary, 3.0 kV; extraction and sampling cone were fixed to four and 35, respectively; source temperature, 120°C; desolation temperature, 400°C [Table 1].

Table 1

MS structural information of 10 constituents in positive ion mode

Quantitative analysis of sesquiterpenes by high performance liquid chromatography-ultraviolet-evaporative light scattering detector

HPLC analysis was performed on Waters system (Milford, MA, USA) equipped with separation module unit and 2487 detector, an Agilent ZORBAX SB-C18 column (4.6 mm *250 mm i.d., 5 μm particles). ELSD analysis was performed with an All tech 2000 ES evaporative light scattering detector. The flow rate of carrier gas (N2) and temperature of drift tube were 3.0 mL/min and 90°C, respectively. The injection volume was 80 μL. A binary gradient elution program was conducted for chromatographic separation with MeCN (A) and ultrapure water (B) as follows: 0–10 min, 42–46% A; 10–20 min, 46–55% A; 20–25 min, 55–60% A; and 25–35 min, 60–75% A. The flow rate and the injection volume were 1.0 mL/min and 10 μL. The column temperature and UV wavelength were set at 30°C and 205 nm.

Ultraviolet and evaporative light scattering detector methodology investigation

Peak areas for the samples and reference substances were integrated by UV detector and ELSD detector, respectively. Limits of detection (LOD) and limits of quantitation (LOQ) were determinates on the basis of their signal to noise (S/N) ratios of 3:1 and 10:1. Precision, the intra-day and inter-day stability, recovery of the two sesquiterpenes were analyzed by UV and ELSD detectors, respectively [Table 2].

Table 2

The Calibration curves, linear range, LOD, LOQ, R2, recovery, and RSD of two sesquiterpenes

Statistical analysis

Experimental results were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance. Statistical significance was determinates as P < 0.05. Each calibration curve was constructed by running standard solutions of six different concentrations. All samples and chromatographic analysis were performed in triplicate.

RESULTS AND DISCUSSION

Result of high performance liquid chromatography time of flight electrospray ionization mass spectrometry on the active extract in Chrysanthemi indici Flos

To develop the quality control, here, the fingerprint of the CTC was established, and 10 constituents including (3R, 5R, 6S, 7S, 10R)-7-(2-hydroxy-2-propyl)-10-methyl-4-methyleneperhy, dronaphthal-ene-3, 5, 6-triol acetone solvate; (+)-edusmance-4, (14)-ene-11, 13-diol; linarin; luteolin; apigenin; tricin; 5, 3’,4’- trimethyl-6,7-dimethoxy flavones; cumambrin A; acacetin; and angeloylcumambrin B were identified by HPLC TOF ESI-MS [Figure 2] and [Table 1]. Among these identified constituents in the CTC, there are six flavones and four sesquiterpenes. The two class constituents have wide distribution in the Composite family such as Taraxacum,[1718] Ligularia,[1920] Sesquiterpene 1 (3R, 5R, 6S, 7S, 10R)-7-(2-hydroxy-2-propyl)-10-methyl-4-methyleneperhy, dronaphthal-ene-3, 5, 6-triol acetone solvate, and sesquiterpene 2 (+)-edusmance-4, (14)-ene-11, 13-diol are eudesmane, sesquiterpene 8 cumambrin A, and sesquiterpene 10 angeloylcumambrin B are guaiane. As terpenoids, cumambrin A, and angeloylcumambrin B have better resolution and contents for quality control [Figure 2].

Quantification the contents of two sesquiterpenes in the active extract in Chrysanthemi indici Flos

The results of the contents of the two sesquiterpenes in CTC analyzed by HPLC-UV-ELSD show that methods were feasible, accurate, and worthy of spreading. UV detector have better sensitivity for LOD and LOQ but baseline drift for edge absorption of 205 nm, while ELSD have good recovery and widely application for eliminating the baseline drift [Table 2]. The contents of two sesquiterpenes decreased with storing years. The contents in 2010 years were lower than that in 2009 years for the terrible harvest quality [Table 3]. Our study offered two different analysis methods for the main sesquiterpenes in CTC. The study also demonstrates that CTC is an active CTC measured by pharmacological action and HPLC TOF ESI-MS.

Table 3

The contents of cumambrin A and angeloylcumambrin B in CTC by UV and ELSD (%, n=3)

CONCLUSIONS

From the results mentioned above, we could see the two main groups including flavones and sesquiterpenes in CTC. It was allowed to propose that the enrichment of flavones and sesquiterpenes in the CTC could influence on the pharmacological action of C. indic extract. However, how does CTC work? And how the simple constituent plays a part in CTC? We did not know, either. Hence, it is necessary to research the connection between the simple constituent and CTC based on the activity of anti-hepatitis B virus and liver protective activity further.