molecules Article
Preparative Separation of Phenolic Compounds from Chimonanthus praecox Flowers by High-Speed Counter-Current Chromatography Using a Stepwise Elution Mode Huaizhi Li 1,2 , Yongqing Zhang 1 , Qian Liu 2 , Changlei Sun 1,2 , Jia Li 1, *, Peng Yang 3 and Xiao Wang 2, * 1 2 3
*
College of Pharmacy, Shandong University of Traditional Chinese Medicine, 4655 Daxue Street, Jinan 250355, Shandong, China;
[email protected] (H.L.);
[email protected] (Y.Z.);
[email protected] (C.S.) Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan 250014, Shandong, China;
[email protected] Kangsen Sanfeng Biological Engineering Technology Co., Jinan 250014, Shandong, China;
[email protected] Correspondence:
[email protected] (X.W.);
[email protected] (J.L.); Tel.: +86-531-8260-5319 (X.W.); +86-531-8962-8081 (J.L.); Fax: +86-531-8296-4889 (X.W.); +86-531-8962-8060 (J.L.)
Academic Editor: Derek J. McPhee Received: 29 June 2016; Accepted: 31 July 2016; Published: 4 August 2016
Abstract: High-speed counter-current chromatography (HSCCC) has been successfully used for the separation of eight compounds from Chimonanthus praecox flowers. Firstly, the crude extract of Chimonanthus praecox flowers was dissolved in a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–H2 O (5:5:3:7, v/v) and divided into two parts: the upper phase (part I) and the lower phase (part II). Then, HSCCC was applied to separate the phenolic acids from part I and part II, respectively. Considering the broad polarity range of target compounds in part I, a stepwise elution mode was established. Two optimal solvent systems of petroleum ether–ethyl acetate–methanol–H2 O–formic acid (FA) (5:5:3:7:0.02, 5:5:4.3:5.7:0.02, v/v) were employed in this separation. Five phenylpropanoids and two flavonoids were successfully separated from 280 mg of part I, including 8.7 mg of 3,4-dihydroxy benzoic acid (a, 95.3% purity), 10.9 mg of protocatechualdehyde (b, 96.8% purity), 11.3 mg of p-coumaric acid (c, 98.9% purity), 12.2 mg of p-hydroxybenzaldehyde (d, 95.9% purity), 24.7 mg of quercetin (e, 97.3% purity), 33.8 mg of kaempferol (f, 96.8% purity), and 24.6 mg of 4-hydroxylcinnamic aldehyde (g, 98.0% purity). From 300 mg of part II, 65.7 mg of rutin (h, 98.2% purity), 7.5 mg of 3,4-dihydroxy benzoic acid (a, 77.4% purity), and 4.7 mg of protocatechualdehyde (b, 81.6% purity) were obtained using the solvent system EtOAc–n-butanol (n-BuOH)–FA–H2 O (4:1:0.5:5, v/v). The structures of the eight pure compounds were confirmed by electrospray ionization-mass spectrometry (ESI-MS), 1 H-NMR and 13 C-NMR. To the best of our knowledge, compounds a–d and f were the first separated and reported from the Chimonanthus praecox flower extract. Keywords: preparative separation; phenolic compounds; Chimonanthus praecox flower; high-speed counter-current chromatography (HSCCC); stepwise elution mode
1. Introduction Chimonanthus praecox (L.) Link (Wintersweet) is a popular potted, garden, cut-flower plant and landscape-design material in most countries for its strong fragrance, long blooming period, and unique flowering time [1–4]. Its flowers are important in traditional medicine in China, and have been used for the treatment of chest tightness, heatstroke, scald, bruise, and particularly as a cough Molecules 2016, 21, 1016; doi:10.3390/molecules21081016
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unique flowering time [1–4]. Its flowers are important in traditional medicine in China, and have been used for the treatment of chest tightness, heatstroke, scald, bruise, and particularly as a cough expectorant [5–7]. Recent Recentresearch research shows phenolic compounds of Chimonanthus expectorant [5–7]. shows thatthat the the phenolic compounds of Chimonanthus praecoxpraecox flower ´ − flower (CPF) extract improve the body’s immune function and efficiently remove O 2 , ‚OH, 1, (CPF) extract improve the body’s immune function and efficiently remove O2 , •OH, 1, 1-diphenyl-21-diphenyl-2-picrylhydrazyl [8,9]. This thata CPF may play a role in anti-aging, picrylhydrazyl (DPPH•) [8,9].(DPPH‚) This implies that CPFimplies may play role in anti-aging, anti-inflammatory, anti-inflammatory, and antioxidation. The main compounds of CPF are volatile oil and However, phenolic and antioxidation. The main compounds of CPF are volatile oil and phenolic compounds. compounds. However, most pharmaceutical research of CPF focused on the volatile oil and few most pharmaceutical research of CPF focused on the volatile oil and few on phenolic compounds on phenolic compounds [10–14]. research Modern showed pharmacological that these [10–14]. Modern pharmacological that theseresearch phenolicshowed compounds have phenolic extensive compounds have extensive activities, such as antioxidant, anti-inflammatory, cardiovascular protection, activities, such as antioxidant, anti-inflammatory, cardiovascular protection, and so on [15–17]. For these and so onestablishing [15–17]. For an these reasons, establishing an efficient and and rapididentify methodthe to separate identify reasons, efficient and rapid method to separate phenolicand compounds the phenolic compounds is needed for studies of the pharmacological and clinical effects of CPF. is needed for studies of the pharmacological and clinical effects of CPF. According silica gelgel column chromatography and and the preparative HPLCHPLC had been Accordingtotothe thereports, reports, silica column chromatography the preparative had used been for thefor separation and purification of several phenolic compounds CPF [18].from However, theseHowever, methods used the separation and purification of several phenolic from compounds CPF [18]. were and theytedious usuallyand offered recoveries. counter-current thesetedious methods were theylow usually offeredHigh-speed low recoveries. High-speedchromatography counter-current (HSCCC) without a solid support matrix is a preparative liquid–liquid chromatography, which could chromatography (HSCCC) without a solid support matrix is a preparative liquid–liquid chromatography, eliminate the irreversible adsorptive loss of samples. loss It enables the separation of the compounds which could eliminate the irreversible adsorptive of samples. It enables thetarget separation of the according to their partition coefficient. With high sample recovery rate, high speed, high loading, target compounds according to their partition coefficient. With high sample recovery rate, high and relative [19,20], HSCCC has been widely used forbeen the separation and of speed, high simplicity loading, and relative simplicity [19,20], HSCCC has widely used forpurification the separation active components from natural products [21–26]. Additionally, it is a big challenge to separate the and purification of active components from natural products [21–26]. Additionally, it is a big challenge target components from the crude extract HSCCC in a by single run, in because of run, the broad polarity to separate the target components from thebycrude extract HSCCC a single because of the range complex stepwise elution modeelution was employed HSCCC to broad and polarity rangecomponents. and complex Therefore, components. Therefore, stepwise mode wasinemployed in address this problem. Finally, eight highly pure compounds were successfully separated and purified HSCCC to address this problem. Finally, eight highly pure compounds were successfully separated from CPF extract (Figure As far (Figure as we know, thefar separation of phenolic compoundsoffrom CPF and the purified from the CPF1).extract 1). As as we know, the separation phenolic by HSCCC isfrom reported for the is first time. here for the first time. compounds CPF here by HSCCC reported
Figure Figure 1. 1. The The chemical chemical structures structuresof ofeight eightphenolic phenoliccompounds compoundsfrom fromChimonanthus Chimonanthuspraecox praecoxflowers. flowers.
2. Results 2. Results and and Discussion Discussion 2.1. 2.1. Selection Selection of of Two-Phase Two-PhaseSolvent SolventSystem System In InHSCCC HSCCCseparation, separation, the the selection selection of of aasuitable suitablesolvent solvent system system isisthe thefirst firstand andmost mostimportant important step. The partition coefficient (K ) is expressed as the mass concentration of the target compound step. coefficient (KDD) is expressed as the mass concentration of the target compound in in stationary phasedivided dividedbybythat thatininthe themobile mobilephase. phase. In In order order to to obtain a rapid thethe stationary phase rapid and and efficient efficient separation, separation,the theoptimal optimalrange rangeof ofKKDD values values is is 0.5 0.5 to to 2.0 2.0 [19,20]. [19,20]. According Accordingto tothe theCPF CPFextract extract profile profileof ofUV UV max max absorption absorption characteristics, characteristics, phenolic phenoliccompounds compounds were wereidentified identifiedto tobe bethe themain maincomponent. component.Considering Consideringthe thepolarity polarityof ofphenolic phenoliccompounds compoundsand andthe the
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experience of of separating separating phenolic phenolic compounds compounds using using HSCCC, HSCCC, several several solvent solvent systems systems composed composed of of experience petroleum ether ether (Pet)–ethyl (Pet)–ethyl acetate acetate (EtOAc)–methanol (EtOAc)–methanol (MeOH)–H (MeOH)–H22O with different different volume volume ratios ratios petroleum O with (5:5:6:4, 5:5:5:5, 5:5:3:7, v/v) were investigated. As shown in Table 1, none of these solvent systems (5:5:6:4, 5:5:5:5, 5:5:3:7, v/v) were investigated. As shown in Table 1, none of these solvent systems were suitable suitable to to separate separate the the target target compounds compounds in in one one step. step. The The crude crude extract extract contains contains complex complex were components, and and their their polarities polarities are are wide, wide, which which brings brings certain certain difficulties their separation. separation. components, difficulties for for their Fortunately, from Table 1, we can see that compounds a–g mainly distributed in the upper Fortunately, from Table 1, we can see that compounds a–g mainly distributed in the upper phase,phase, while whileofmost of the impurities andcompound target compound were allocated to thephase lowerinphase in the solvent most the impurities and target h were hallocated to the lower the solvent system system Pet–EtOAc–MeOH–H 2O (5:5:3:7, v/v). By this token, the solvent system could be used to Pet–EtOAc–MeOH–H 2 O (5:5:3:7, v/v). By this token, the solvent system could be used to pretreat the pretreat the crude extract to achieve thepurification. preliminaryTherefore, purification. the crude extracts crude extract to achieve the preliminary theTherefore, crude extracts were dealt withwere the dealt with the solvent system Pet–EtOAc–MeOH–H 2Oand (5:5:3:7, v/v) and divided into two parts: the solvent system Pet–EtOAc–MeOH–H O (5:5:3:7, v/v) divided into two parts: the upper phase 2 upper phase (part I) and the lower phase (part II), which were used for further separation by (part I) and the lower phase (part II), which were used for further separation by HSCCC. The HPLC HSCCC. The HPLC chromatograms are shown in Figure 2. chromatograms are shown in Figure 2.
Figure 2. 2. HPLC Chimonanthus praecox flower (CPF) Figure HPLC chromatograms chromatograms of of part part I, part II, and the Chimonanthus extract. Pet: petroleum ether; EtOAc: ethyl acetate; MeOH: methanol. a. 3,4-dihydroxy benzoic acid, extract. b. protocatechualdehyde, protocatechualdehyde, c. c. p-coumaric p-coumaric acid, acid, d. d. p-hydroxybenzaldehyde, e. quercetin, f. kaempferol, b. g. 4-hydroxylcinnamic 4-hydroxylcinnamic aldehyde, aldehyde, h. h. rutin. rutin. g. Table1. coefficient (K (KDD) of the target compounds in several solvent systems. Table 1. Partition coefficient Sample Sample Crude extract a
Crude extract a Part I Part I
Part II Part II
Solvent System (v/v) Solvent System (v/v) Pet–EtOAc–MeOH–H2O 5:5:6:4 Pet–EtOAc–MeOH–H 5:5:5:5 Pet–EtOAc–MeOH–H2O 2 O 5:5:6:4 Pet–EtOAc–MeOH–H Pet–EtOAc–MeOH–H2O O 5:5:5:5 2 5:5:3:7 Pet–EtOAc–MeOH–H 5:5:3:7 2O–FA 5:5:4:6:0.02 Pet–EtOAc–MeOH–H 2O Pet–EtOAc–MeOH–H2O–FA 5:5:4:6:0.02 Pet–EtOAc–MeOH–H 2 O–FA5:5:3:7:0.02 Pet–EtOAc–MeOH–H 5:5:3:7:0.02 2 O–FA 2O–FA 5:5:4.2:5.8:0.02 Pet–EtOAc–MeOH–H Pet–EtOAc–MeOH–H2O–FA 5:5:4.2:5.8:0.02 2 O–FA5:5:4.3:5.7:0.02 Pet–EtOAc–MeOH–H Pet–EtOAc–MeOH–H 5:5:4.3:5.7:0.02 2 O–FA EtOAc–EtOH–FA–H 2O 4:1:0.1:5 EtOAc–EtOH–FA–H O 4:1:0.1:5 EtOAc–n-BuOH–FA–H22O 4:1:0.1:5 EtOAc–n-BuOH–FA–H2 O 4:1:0.1:5 4:1:0.5:5 EtOAc–n-BuOH–FA–H2O EtOAc–n-BuOH–FA–H2 O 4:1:0.5:5
a a 0.11 0.21 0.11 0.79 0.21 0.79 0.17 0.17 0.47 0.47 0.23 0.23 0.16 0.16 – – – – – –
b b 0.37 0.58 0.37 1.32 0.58 1.32 0.25 0.25 0.59 0.59 0.37 0.37 0.18 0.18 – – – – – –
Partition Coefficient (KD) bb Partition Coefficient (KD ) c d e f c d e f 2.53 1.97 1.03 8.62 3.01 >10 2.53 2.79 1.97 1.38 1.03 8.62 5.36 >10 3.01 5.21 2.79 3.01 1.38 >10 5.36 0.99 5.21 1.26 3.01 >10 0.43 1.77 0.43 1.42 0.99 1.76 1.26 1.77 0.98 2.56 0.98 0.94 1.42 0.97 1.76 2.56 0.28 1.35 0.28 0.64 0.94 0.92 0.97 1.35 0.25 1.12 0.25 0.64 0.92 1.12 – – – – – – – – – – – – – – – – – – – – – – – –
g g >10 >10 >10 >10 >10 >10 2.37 2.37 3.62 3.62 1.83 1.83 1.51 1.51 – – – – – –
h h
