molecules Article
An Efficient Method for the Preparative Isolation and Purification of Flavonoids from Leaves of Crataegus pinnatifida by HSCCC and Pre-HPLC Lei Wen 1 , Yunliang Lin 2 , Ruimin Lv 2 , Huijiao Yan 2 , Jinqian Yu 2 , Hengqiang Zhao 2 , Xiao Wang 2 and Daijie Wang 2, * 1 2
*
College of Pharmacy, Shandong University of Traditional Chinese Medicine, 4655 Daxue Road, Jinan 250355, China;
[email protected] Shandong Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan 250014, China;
[email protected] (Y.L.);
[email protected] (R.L.);
[email protected] (H.Y.);
[email protected] (J.Y.);
[email protected] (H.Z.);
[email protected] (X.W.) Correspondence:
[email protected]; Tel.: +86-531-8260-5319; Fax: +86-531-8296-4889
Academic Editor: Stefan Berger Received: 8 March 2017; Accepted: 3 May 2017; Published: 9 May 2017
Abstract: In this work, flavonoid fraction from the leaves of Crataegus pinnatifida was separated into its seven main constituents using a combination of HSCCC coupled with pre-HPLC. In the first step, the total flavonoid extract was subjected to HSCCC with a two-solvent system of chloroform/methanol/ water/n-butanol (4:3:2:1.5, v/v), yielding four pure compounds, namely (–)-epicatechin (1), quercetin-3-O-(2,6-di-α-L-rhamnopyranosyl)-β-D-galactopyranoside (2), 400 -O-glucosylvitexin (3) and 200 -O-rhamnosylvitexin (4) as well as a mixture of three further flavonoids. An extrusion mode was used to rapidly separate quercetin-3-O-(2,6-di-α-L-rhamnopyranosyl)-β-D-galactopyranoside with a big KD -value. In the second step, the mixture that resulted from HSCCC was separated by pre-HPLC, resulting in three pure compounds including: vitexin (5), hyperoside (6) and isoquercitrin (7). The purities of the isolated compounds were established to be over 98%, as determined by HPLC. The structures of these seven flavonoids were elucidated by ESI-MS and NMR spectroscopic analyses. Keywords: Crataegus pinnatifida leaves; flavonoids; HSCCC and pre-HPLC combination; extrusion mode
1. Introduction Crataegus pinnatifida, Rosaceae family, is widely spread in Northern China and is considered as a famous medicinal and edible plant. The leaves of C. pinnatifida, a famous folk medicine, have been used clinically for dredging the meridian, lowering blood lipid levels, activating blood flow and removing blood stasis [1]. So far, many compounds including flavonoids, triterpenoids, steroids, monoterpenoids, sesquiterpenoids, lignans, hydroxycinnamic acids, organic acids and nitrogen-containing compounds have been isolated and identified from C. pinnatifida. Among them, the flavonoids are considered to be the major bioactive constituents with many pharmacological actions, such as treating fatty livers, showing antioxidant activity, and exhibiting a protective effect on cell toxicity and protease inhibitory activities [2–6]. Previously, flavonoids from the leaves of C. pinnatifida were isolated and purified by conventional techniques, such as polyamide chromatography, silica gel column chromatography and sephadex LH-20, which were a waste organic solvent, tedious, time-consuming and often resulted in irreversible adsorption of the compounds [7–14]. Thus, effective separation methods for flavonoids from the leaves of C. pinnatifida are of great importance.
Molecules 2017, 22, 767; doi:10.3390/molecules22050767
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Molecules 2017, 22, 767
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Molecules 2017, 22, 767
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irreversible adsorption of the compounds [7–14]. Thus, effective separation methods for flavonoids from the leaves of C. pinnatifida are of great importance. High-speed counter-current counter-current chromatography chromatography (HSCCC) (HSCCC) has has now now been been widely widely applied applied in in the the High-speed separationofof various compounds, as a liquid-liquid partition chromatography [15]. speaking, Generally separation various compounds, as a liquid-liquid partition chromatography [15]. Generally speaking, HSCCC needs no solid support, which thathigh it possesses high loading capacity, HSCCC needs no solid support, which indicates thatindicates it possesses loading capacity, low economic lowand economic cost and no irreversible of samples. Therefore, HSCCC been cost no irreversible adsorption of samples.adsorption Therefore, HSCCC has been developed to be anhas efficient developed to be an efficient tool for theofpurification and separation tool for the purification and separation various samples [16–22]. of various samples [16–22]. Ina aprevious previous study, three flavonoids the leaves of C. pinnatifida were using isolated using In study, three flavonoids fromfrom the leaves of C. pinnatifida were isolated HSCCC HSCCCwith coupled with macroporous resin and pre-HPLC In the thatsolvent work, system the solvent coupled macroporous resin and pre-HPLC [23]. In that[23]. work, was system selectedwas by selected by analytical HSCCC, and did not measure the K D -values. Furthermore, the flavonoids analytical HSCCC, and did not measure the KD -values. Furthermore, the flavonoids from the leaves from the leaves were of C. partial, pinnatifida were partial, 20% ethanol of D101resin. macroporous of C. pinnatifida acquired by 20%acquired ethanolby elution of D101elution macroporous In this resin.aInnew thistwo-phase work, a new two-phase system HSCCC was developed for and the purification work, solvent system solvent of HSCCC was of developed for the purification separation and separation of flavonoids from the leaves Furthermore, of C. pinnatifida. an was extrusion mode was of flavonoids from the leaves of C. pinnatifida. an Furthermore, extrusion mode used to rapidly used tocompounds rapidly separate compounds withmixture big KDwas -values. The mixture was furtherFinally, purified by separate with big KD -values. The further purified by pre-HPLC. seven pre-HPLC.were Finally, seven flavonoids were by separated by ESI-MSanalyses. and NMR spectroscopic flavonoids separated and elucidated ESI-MSand andelucidated NMR spectroscopic The structures analyses. The structures of these of these compounds are shown in compounds Figure 1. are shown in Figure 1. OH
OH OH
OH
O
HO
O
HO OH
HO R
OH
OH 1
R
OH O
OH O
O
2 R=O-β-D-Gal-(2,6-di-α-L-Rha) 6 R=O-β-D-Gal 7 R=O-β-D-Glc
3 R=β-D-Glc(4-1)-β-D-Glc 4 R=β-D-Glc-2''-α-L-Rha 5 R=β-D-Glc
Figure Figure1.1.Structures Structuresof ofthe thecompounds compoundsfrom fromC. C.pinnatifida. pinnatifida.
Resultsand andDiscussion Discussion 2.2.Results 2.1. 2.1.Optimization OptimizationofofHSCCC HSCCCConditions Conditions The The separation separation of of the the seven seven flavonoids flavonoids from from the the leaves leaves of of C. C. pinnatifida pinnatifida was was carried carried out out by by HSCCC. A successful separation of the target compounds using HSCCC required a careful search HSCCC. A successful separation of the target compounds using HSCCC required a careful search for foraasuitable suitabletwo-phase two-phasesolvent solventsystem systemtotoprovide providean anideal idealrange rangeofofKD K-values. D-values. According According to tothe the structural characteristics of flavonoids, three series of solvent systems including n-hexane/ethyl structural characteristics of flavonoids, three series of solvent systems including n-hexane/ethyl acetate/methanol/water, acetate/methanol/water, n-hexane/ethyl n-hexane/ethyl acetate/ethanol/water acetate/ethanol/water and and chloroform/ethanol/water chloroform/ethanol/water were were designed designedon onthe thebasis basisofofprevious previousresearch research[24]. [24].The TheKKDD-values -valuesof ofthe thetarget targetcompounds compoundsin inthe thethree three series seriesof ofsolvent solventsystems systemswere weredetected detectedand andcalculated, calculated,asasshown shownininTable Table1.1. Table Table1.1.The TheKKDD-values -valuesof offlavonoids flavonoidsfrom fromthe theleaves leavesofofC. C.pinnatifida. pinnatifida. Solvent System
Solvent System
n-Hex–EtOAc–MeOH–H2O (1:5:1:5, v/v) n-Hex–EtOAc–EtOH–H 2O (1:1.6:1:1.6, v/v) n-Hex–EtOAc–MeOH–H2 O (1:5:1:5, v/v) 2O (4:3:2, v/v) CHCl3–MeOH–H n-Hex–EtOAc–EtOH–H O (1:1.6:1:1.6, v/v) 2 3–MeOH–H2O–n-BuOH (4:3:2:0.5, v/v) CHCl CHCl –MeOH–H O (4:3:2, v/v) 3 2 CHCl3–MeOH–H2O–n-BuOH (4:3:2:1, v/v) CHCl3 –MeOH–H 2 O–n-BuOH (4:3:2:0.5, v/v) CHCl3–MeOH–H2O–n-BuOH (4:3:2:1.5, v/v)
CHCl3 –MeOH–H2 O–n-BuOH (4:3:2:1, v/v) CHCl3 –MeOH–H2 O–n-BuOH (4:3:2:1.5, v/v)
1 14.43 0.12 4.43 7.62 0.12 5.13 7.62 3.88 5.13 2.15
3.88 2.15
2 2 0.40 30 30 >30 26.49 >30 18.91
26.49 18.91
KD-Values KD -Values 3 4 5 6 3 0.41 0.12 4
