Mikania micrantha (M. micrantha), a perennial vine (Asteraceae family), is native from South America, where it is a weed of minor importance [1]. However, the weed is one of the worst invasive alien species in tropical and subtropical areas [2]. M. micrantha has been present in South China since 1980s, and in recent years, it has caused more and more damages to ecosystems [3, 4]. However, M. micrantha has been used as folk medicine to treat asthma, bronchitis, coughing and snake bites in some places. Many studies indicated that this weed had biological activities, such as anti-inflammatory, analgesic, antimicrobial, and contained several medicinal-valued compounds mainly sterols, terpenoids and flavonoids [5-8]. Flavonoids are claimed to possess various pharmacology effects. They are major functional components of many herbs for medical use [9, 10]. So how to effectively separate these virtual components becomes especially crucial.
High speed countercurrent chromatography (HSCCC) is a support-free liquid-liquid partition chromatographic technique which provides an advantage over the conventional column chromatography by eliminating the irreversible adsorption [11]. It has been widely used as a robust preparative technique [12]. As a method easy to be scaled up, HSCCC has been widely used in the separation of natural products [13-18]. However, most of the time, HSCCC cannot directly separate complicated compounds due to its limited column efficiency. Macroporous resin adsorption technology has recently drawn more attention in pharmaceutical applications and natural products separation field for pretreatment due to its low-cost, high-efficiency, easy-recycling and simple scaling-up performance [19-21]. Macroporous resin can be reused for thousands of times. So this technique is friendly to the environment [22]. It has been reported that macroporous resin could separate several flavonoids effectively [23]. Therefore, macroporous resin adsorption technique combined with HSCCC might be a suitable method for the separation and purification of flavonoids from natural products [24-26].
The present paper aims to establish an effective preparative separation and purification method for the four flavonoids, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, luteoloside and astragalin from M. micrantha by using macroporous resin combined with HSCCC.
M. micrantha was supplied by the Institute of Plant Protection, Chinese Academy of Agricultu-ral Sciences. All solvents used in HSCCC separation were of analytical grade (Tianjin Damao Chemical Reagent Factory, China). Macroporous resins D4020 and AB-8 were purchased from Chemical Plant of Nankai University (Tianjin, China).
The HSCCC instrument 1 (Beijing Institute of New Technology Application, Beijing, China) was a Model GS10AB multilayer coil planet centrifuge equipped with a polytetrafluoroethylene (PTFE) multilayer coil of 110 m×1.6 mm i. d., with a total capacity of 220 mL. The β values (β=r/R, where r is the distance from the holder shaft to the coil, and R is the distance between the holder axis and central axis of the planet centrifuge or the rotation radius) of the coil varied from 0.5 (internal terminal) to 0.75 (external terminal).
The HSCCC instrument 2 was used with a customized multilayer coil planet centrifuge. The apparatus was consisting of type-J coil planet centrifuge, equipped with a separation column and a counter weight in symmetrical position at a distance of 10 cm from the central axis of the centrifuge. In this planetary centrifuge, the separation column revolves around the central axis of the centrifuge while it synchronously rotates around its own axis at the same direction. The separation column was made up of a spiral tube purchased from CC Biotech, USA. It has four spiral interwoven grooves, each 2.8 mm wide and 5 cm deep with four transfer radial grooves. PTFE tubing of 1.6 mm i. d. (SW14, Zeus Industrial Products, USA) was flat-twisted and accommodated tightly into the spiral tube support by squashing it with a tool which fits to the radial grooves. There were 10 spiral layers and the total capacity was about 80 mL.
Although the revolution speed of the apparatus could be regulated with a speed controller in a range of 0 to 1 000 r/min, an optimum speed of 800 r/min was used in the present study.
The analytical high-performance liquid chromatography (HPLC) equipment used a Shimadzu LC-20AVP system equipped with two LC-20AT solvent delivery units, an SPD-M20AVP UV-VIS photodiode array detector (DAD) system, a Model 7725 injection valve with a 20 μ L loop and an auto-sampler, an SCL-20AVP system controller, and a Class-VP-LC work station (Shimadzu, Kyoto, Japan).
The dried whole herb M. micrantha was pulverized by a shredding machine, and 50 g of powder was extracted with 250 mL of 80% (v/v) ethanol for 150 min three times. Then, the extract was combined, filtered and concentrated under reduced pressure by rotary evaporation at 55 ℃.
Before the experiment, the macroporous resin was soaked with 95% (v/v) ethanol for 24 h to remove some impurities. Then the macroporous resin was washed by 5% (v/v) HCl solution, deionized water, 5% (v/v) NaOH solution, deionized water respectively to remove the monomers and porogenic agents trapped inside the pores during the synthesis process.
The resin-based column chromatography was performed on a low-pressure glass chromatographic column (150 cm×100 mm i. d.) filled with macroporous resin. The concentration of the sample solution was 5 g/L, the volume of the sample solution is 8 bed volume (BV), flow rate is 4 mL/min, and the volume of the eluent is 2 BV.
The solvent system composed of hexane/ethyl acetate/methanol/water (HEMW, 5 : 5 : 5 : 5 and 6 : 4 : 5 : 5, v/v), ethyl acetate/methanol/water (10 : 1 : 10, v/v) and n-butanol/acetic acid/water (4 : 1 : 5, v/v) was used for HSCCC separation. The preparation of each two-phase solvent system was performed in a separatory funnel according to the volume ratios and equilibrated after shaking at room temperature. The upper phase and lower phase were then separated and degassed by ultrasonic for 30 min before use.
The sample solution for HSCCC was prepared by dissolving the sample after macroporous resin column chromatography 300 mg in 4 mL solvent mixture, which was composed of 2 mL upper phase and 2 mL lower phase.
HSCCC separation was performed as follows: the separation column of HSCCC was entirely filled with the stationary phase (the upper phase) by constant flow at 5 mL/min. The separation temperature was room temperature (25 ℃). Then, the lower phase was pumped into multilayer coiled column at a flow rate of 1.0 mL/min while the apparatus was run at a revolution speed of 800 r/min. After hydrodynamic equilibrium was established, the sample solution was injected through the injection valve as indicated by a clear mobile phase eluting outlet. The effluent was continuously monitored with a UV-Vis detector at 254 nm. Each peak fraction was collected according to the chromatogram. All the fractions were detected by HPLC.
HPLC conditions were as follows: Apollo C18 column (150 mm×4.6 mm i. d., 5 μ m); gradient elution was performed using eluent A (MeOH) and eluent B (0.05% (v/v) H3PO4) with the following linear gradient combinations: 0-10 min, 25% A-37% A; 10-35 min, 37% A-42% A; 35-40 min, 42% A-62% A; 40-55 min, 62% A-85% A; 55-60 min, 85% A-95% A; 60-70 min, 95% A; 70-80 min, 25% A.
The HPLC chromatogram of crude sample and the chemical structures of the four flavonoids are shown in Fig. 1. Four standards (quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, luteoloside and astragalin) were used to identify the target compounds.
As seen in the HPLC chromatogram (Fig. 2), the flavonoids were present from 20 min to 40 min and there were some impurities which might influence the HSCCC separation followed. To improve the preparative separation efficiency, remove the impurities and enrich flavonoids, the macroporous resin column chromatography was used.
It is a critical step to select the suitable macroporous resin for efficient enrichment of flavonoids. Based on the previous work in our laboratory [23], we compared two kinds of macroporous resins D4020 and AB-8.
In the experiment, water and ethanol solutions of different concentrations (from 0 to 100%, v/v) were used to elute the column in series. According to the polarity of flavonoids, finally 50% (v/v) ethanol solution was selected as eluate based on the polarity of flavonoid and HPLC detection. The results showed that the faction between 10 min and 50 min eluted by AB-8 using 50% (v/v) ethanol was clearer, and the amount of impurities was less. So AB-8 was a suitable resin for flavonoid enrichment and impurity removal from M. micrantha (Fig. 3).
In order to get a rapid separation of the aim flavonoids, firstly we tried a common solvent system hexane/ethyl acetate/methanol/water (5 : 5 : 5 : 5, 6 : 4 : 5 : 5, v/v) for separating flavonoids. The results are shown in Fig. 4. Although these two solvent systems provided a suit-able retention times, they showed poor peak resolution and the sample was eluted by the mobile phase at one time. It indicated that the compounds could not be well retained on the stationary phase. The polarities of the solvent system should be increased according to the results. So the ethyl acetate/methanol/water (10 : 1 : 10, v/v) solvent system was selected. The results are shown in Fig. 4c. The fractions were analyzed by HPLC and the results are shown in Fig. 5.
Considering the polarities of flavonoids, peak 2 (Fig. 4c) was the target compound astragalin, but it had a low response value in HPLC (Fig. 5). Meanwhile peak 1 contained many compounds, most compounds were eluted by mobile phase which have large polarity at one time. Due to the above results, the difference between the upper phase and the lower phase should be reduced. The solvent system composed of n-butanol/acetic acid/water (4 : 1 : 5, v/v) was finally selected for the further experiments.
As shown in Fig. 6a, the solvent system of n-butanol/acetic acid/water (4 : 1 : 5, v/v) was used and the elution mode was from tail to head. Four peaks of target compounds were obtained in one run. Each peak was analyzed by HPLC and the chromatograms are shown in Fig. 6b. The first fraction is the unknown compounds plus impurities. Peaks 1, 2, 3 and 4 are quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, luteoloside and astragalin with the purities of 90.12%, 98.55%, 98.33% and 99.23% respectively.
In this work, an effective method of separating flavonoids from M. micrantha was built. Four flavonoids quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, luteoloside and astragalin were resolved well in one run from an ethanol aqueous extraction of this weed by using AB-8 resin-based column chromatography combined with HSCCC using solvent system comprised of n-butanol/acetic acid/water (4 : 1 : 5, v/v). This method would pave the pathway to develop more separation technology for the systematic separation of other bioactive compounds from invasive plants.
2017,
Vol. 35 Issue (3): 302-307
PDF
2017,
Vol. 35 Issue (3): 302-307
PDF