TY - JOUR
AU - Fu,, Yu-Jie
AB - Abstract Morus alba L. is a medicinal plant that contains a high amount of caffeoylquinic acids such as 3-caffeoylquinic acid (3-CQA), 5-caffeoylquinic acid (5-CQA) and 4-caffeoylquinic acid (4-CQA). This study aimed to establish a fast and efficient method for separating caffeoylquinic acids from mulberry leaves by using high-speed countercurrent chromatography coupled with macroporous resin. D101 resin showed better adsorption and desorption capacity for three caffeoylquinic acids among six macroporous resin adsorbents. The contents of 3-CQA, 5-CQA and 4-CQA reached for 4.77%, 18.95% and 9.84% through one cycle of D101 resin, which were 3.13-fold, 4.57-fold and 4.78-fold more than those in crude extracts, respectively. With a two-phase solvent system of ethyl acetate-water (1:1, V/V), >93% purity of target compounds were obtained in one cycle during 150 min with the recovery yields of 80.59%, 99.56% and 94.21% for 3-CQA, 5-CQA and 4-CQA, respectively. The structural identification of target compounds was carried out by ESI-MS, 1H NMR and 13C NMR spectra. The present result represented an easy and efficient separation strategy for the utilization of mulberry resource. Introduction Morus alba L. is widely distributed throughout Asia and have been used not only as a silkworm feed and food additive but also as a traditional Chinese medicine for long period. Its leaves possess more interesting biological activities such as suppressing hyperglycemia (1, 2), anti-atherogenic (3), antioxidant (4), antineoplastic (5), anti-inflammatory (6, 7) and anti-obesity (8) properties. Phytochemical studies indicated that the mulberry leaves were rich in flavones and caffeoylquinic acids (9, 10), including 3-caffeoylquinic acid (3-CQA), 5-caffeoylquinic acid (5-CQA) and 4-caffeoylquinic acid (4-CQA) (9). Among these, caffeoylquinic acids exhibited notable antioxidant (11), hypocholesterolemic (12), chondroprotective, antinociceptive (13) and antihypertensive effects (14). Furthermore, caffeoylquinic acids can be used as natural α-glucosidase (15), PTP1B (15, 16) and PD-1/PD-L1 (17) inhibitors for diabetes, obesity and cancer treatments. Recently, few reports indicated that the caffeoylquinic acids can be obtained by artificial synthesis to meet the market demand, which is mainly based on extraction and separation from plants. However, it is difficult to separate caffeoylquinic acids from plants because of their similar structure and polarity. Therefore, their separations have become of great importance, which would promote the maximum utilization of renewable mulberry resources. Conventional methods such as silica gel column chromatography and liquid–liquid extraction have many disadvantages, such as irreversible adsorption of sample components, laboriousness, low production and high cost (18, 19). In recent years, macroporous resin have been widely used for separation and enrichment of natural production plant resources due to their good performances, simple procedure, better selectivity, low cost and reusability (20–23). So far, macroporous resins have been widely used in the separation and enrichment of phytochemicals, such as flavone aglycones (21), stilbenes (22), isoflavones (23, 24), paclitaxel (25) and anthocyanins (22, 26). In addition, high-speed countercurrent chromatography (HSCCC) (27), which was a nonsolid support in liquid–liquid chromatography separation technique that eliminating the risk of irreversible adsorption of sample components. HSCCC has great advantages, such as rapid separation, low solvent consumption, high recovery, low risk of sample denaturation, high repeatability and large injection volume (18, 19, 28). Hence, the development of an efficient and inexpensive technology for preparative separation of caffeoylquinic acids from mulberry leaves with HSCCC is urgently needed. According to our knowledge, preparative separation of caffeoylquinic acids from mulberry with HSCCC coupled with macroporous resin has not been reported yet. The aim of this research is to establish a fast and efficient method for separation and purification of three caffeoylquinic acids from mulberry leaves using HSCCC coupled with macroporous resin. Therefore, the resin was selected and relevant process parameters were optimized. The best HSCCC separation conditions were investigated through the effects of two-phase solvent system and sample loading. Finally, the chemical structures of the separated compounds were confirmed by Electrospray Ionization Mass Spectrometry (ESI-MS)-, 1H Nuclear magnetic resonance (NMR) and 13C NMR. Experimental Materials and reagents 3-CQA, 5-CQA and 4-CQA (⩾98%) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (J&K Scientific Ltd, Beijing, China) were used for High performance liquid chromatography (HPLC) analysis. Solvents were analytical grade (Tianjin Chemical Reagents Company, Tianjin, China). Ultra-pure water was obtained from a Milli-Q water purification system (Millipore, Boston, MA, USA). Six macroporous resins including NKA-9, NKA-II, HPD826, AB-8, D101 and HPD100 were purchased from Bon Chem (Hebei, China). Apparatus HSCCC system was used by TBE-300C (TAUTO, Shanghai, China) with three multilayer coil separation columns (internal diameter of tubing, 1.9 mm; total volume, 300 mL) and a 20 mL manual sample loop. The revolution radius was 5 cm, and the β-values of the multilayer coil ranged from 0.5 (at the internal terminal) to 0.8 (at the external terminal). Apparatus revolution speed was regulated with a speed controller within the range of 0–1000 rpm. Preparation of mulberry leaves sample solutions Mulberry leaves were collected from experimental forest farm in the Northeast Forestry University. The leaves were cleaned by distilled water and dried in the shade at room temperature. Afterwards, the dried samples were pulverized, sieved (40 mesh) and stored in a freezer at 4°C until analyzed. The leaves (1 kg) were extracted three times (each for 45 min) with 4 L of 50% ethanol (v/v) by an ultrasonic bath (Kunshan Ultrasonic Instrument Co. Ltd, China) at room temperature. The combined filtrates were concentrated to alcohol free, then suspended in water and adjusted pH to 4. And the extraction yield value can be calculated as extraction yield = peak area of each extraction/initial peak area of the sample (Figure 1). Finally, the sample solution was extracted with n-butanol, concentrated and dissolved in water for the resin adsorption and desorption tests. The concentration of 3-CQA was 0.47 mg/mL, 5-CQA was 1.29 mg/mL and 4-CQA was 0.65 mg/mL. Figure 1 Open in new tabDownload slide (A) HSCCC chromatogram for three target caffeoylquinic acids. (B) HPLC chromatography of crude extract (a) and three HSCCC fractions (b, 3-CQA; c, 5-CQA; d, 4-CQA). 1 represents 3-CQA; 2, 4-CQA; 3, 5-CQA. HSCCCconditions: biphasic system: ethyl acetate: water (50:50); revolution speed: 900 rpm; detection wavelength: 325 nm; flow rate: 2 mL/min; separation temperature: 25°C. Figure 1 Open in new tabDownload slide (A) HSCCC chromatogram for three target caffeoylquinic acids. (B) HPLC chromatography of crude extract (a) and three HSCCC fractions (b, 3-CQA; c, 5-CQA; d, 4-CQA). 1 represents 3-CQA; 2, 4-CQA; 3, 5-CQA. HSCCCconditions: biphasic system: ethyl acetate: water (50:50); revolution speed: 900 rpm; detection wavelength: 325 nm; flow rate: 2 mL/min; separation temperature: 25°C. Table I Partition coefficients (K) and separation factors (α) measured for the target compounds in different solvent systems Solvent systems Volume ratio K1a K2a K3a α1 α2 Ethyl acetate: water 1:1 0.2536 0.5604 0.3062 2.2098 1.8302 Ethyl acetate: n-butanol: water 4:1:5 0.4591 0.1676 0.0904 2.7393 1.8540 Ethyl acetate: n-butanol: water 3:2:5 0.8123 0.2571 0.1418 3.1595 1.8131 Ethyl acetate: n-butanol: water 2:3:5 1.1049 0.2738 0.1663 4.0354 1.6464 Ethyl acetate: n-butanol: water 1:4:5 0.8779 0.2581 0.1490 3.4014 1.7322 n-butanol: water 1:1 0.7539 0.2233 0.1273 3.3762 1.7541 Ethyl acetate: n-butanol: water 1:3:4 1.1207 0.2892 0.1924 3.8752 1.5031 Ethyl acetate: acetic acid: water 4:1:5 1.4344 0.2031 0.1537 7.0625 1.3214 Ethyl acetate: acetic acid: water 3:1:7 0.3211 0.0611 0.0466 5.2553 1.3112 Solvent systems Volume ratio K1a K2a K3a α1 α2 Ethyl acetate: water 1:1 0.2536 0.5604 0.3062 2.2098 1.8302 Ethyl acetate: n-butanol: water 4:1:5 0.4591 0.1676 0.0904 2.7393 1.8540 Ethyl acetate: n-butanol: water 3:2:5 0.8123 0.2571 0.1418 3.1595 1.8131 Ethyl acetate: n-butanol: water 2:3:5 1.1049 0.2738 0.1663 4.0354 1.6464 Ethyl acetate: n-butanol: water 1:4:5 0.8779 0.2581 0.1490 3.4014 1.7322 n-butanol: water 1:1 0.7539 0.2233 0.1273 3.3762 1.7541 Ethyl acetate: n-butanol: water 1:3:4 1.1207 0.2892 0.1924 3.8752 1.5031 Ethyl acetate: acetic acid: water 4:1:5 1.4344 0.2031 0.1537 7.0625 1.3214 Ethyl acetate: acetic acid: water 3:1:7 0.3211 0.0611 0.0466 5.2553 1.3112 aK1 represents 3-CQA; K2, 5-CQA; K3, 4-CQA. Open in new tab Table I Partition coefficients (K) and separation factors (α) measured for the target compounds in different solvent systems Solvent systems Volume ratio K1a K2a K3a α1 α2 Ethyl acetate: water 1:1 0.2536 0.5604 0.3062 2.2098 1.8302 Ethyl acetate: n-butanol: water 4:1:5 0.4591 0.1676 0.0904 2.7393 1.8540 Ethyl acetate: n-butanol: water 3:2:5 0.8123 0.2571 0.1418 3.1595 1.8131 Ethyl acetate: n-butanol: water 2:3:5 1.1049 0.2738 0.1663 4.0354 1.6464 Ethyl acetate: n-butanol: water 1:4:5 0.8779 0.2581 0.1490 3.4014 1.7322 n-butanol: water 1:1 0.7539 0.2233 0.1273 3.3762 1.7541 Ethyl acetate: n-butanol: water 1:3:4 1.1207 0.2892 0.1924 3.8752 1.5031 Ethyl acetate: acetic acid: water 4:1:5 1.4344 0.2031 0.1537 7.0625 1.3214 Ethyl acetate: acetic acid: water 3:1:7 0.3211 0.0611 0.0466 5.2553 1.3112 Solvent systems Volume ratio K1a K2a K3a α1 α2 Ethyl acetate: water 1:1 0.2536 0.5604 0.3062 2.2098 1.8302 Ethyl acetate: n-butanol: water 4:1:5 0.4591 0.1676 0.0904 2.7393 1.8540 Ethyl acetate: n-butanol: water 3:2:5 0.8123 0.2571 0.1418 3.1595 1.8131 Ethyl acetate: n-butanol: water 2:3:5 1.1049 0.2738 0.1663 4.0354 1.6464 Ethyl acetate: n-butanol: water 1:4:5 0.8779 0.2581 0.1490 3.4014 1.7322 n-butanol: water 1:1 0.7539 0.2233 0.1273 3.3762 1.7541 Ethyl acetate: n-butanol: water 1:3:4 1.1207 0.2892 0.1924 3.8752 1.5031 Ethyl acetate: acetic acid: water 4:1:5 1.4344 0.2031 0.1537 7.0625 1.3214 Ethyl acetate: acetic acid: water 3:1:7 0.3211 0.0611 0.0466 5.2553 1.3112 aK1 represents 3-CQA; K2, 5-CQA; K3, 4-CQA. Open in new tab Enrichment of caffeoylquinic acids by macroporous resin The static absorption and desorption tests were accomplished as follows: preweighed amounts of hydrated resins (equal to 1.0 g dry resin) were added to 100 mL conical flasks containing 15 mL of sample solution. The flasks were sealed tightly and shaken at 100 rpm for 5 h in a constant temperature oscillator at 25°C. After reaching the adsorption equilibrium, the residual solutions were then filtered and the concentrations of three caffeoylquinic acids were analyzed by HPLC. Then the resins were desorbed with 15 mL 50% ethanol solution and shaken (100 rpm) for 5 h at 25°C. Desorption solutions were then separated from the resins and analyzed by HPLC. The dynamic breakthrough curves were constructed in a glass column (10 × 500 mm) filled with the selected resin (5.0 g dry weight) at a bed volume (BV) of 24 mL. The sample solution was flowed through the resin column at the rate of 1.0 BV/h. During dynamic adsorption, the concentrations of three caffeoylquinic acids in the eluents collected at 1/3 BV intervals were analyzed by HPLC. After reaching the adsorption equilibrium, the column was first washed thoroughly with deionized water, and then eluted with 2 BV different concentrations of ethanol (10%, 20%, 30% and 40%) at a constant flow rate of 1 BV/h. The concentrations of three caffeoylquinic acids in desorption solutions were determined by HPLC. Finally, the resin column, after adsorption equilibrium, was successively eluted by 10% ethanol–water solution at different column volumes. Each part of desorption solutions was measured using HPLC. Laboratory preparative-scale enrichment by D101 resin was performed under the optimized parameters to obtain enriched sample for further the preparative separation caffeoylquinic acids by HSCCC. Selection of two-phase solvent systems The two-phase solvent system was selected by measuring the partition coefficient (K) and separation factor (α) of each target compound, which was analyzed by HPLC as previously reported (29, 30). A suitable amount of the D101-enriched resin sample was added into a 25 mL separatory funnel, and 10 mL of each phase of a pre-equilibrated two-phase solvent system was added, shaken vigorously and equilibrated for 20 min. Then the upper and lower phases were separated and evaporated to dryness. Subsequently, the residues were dissolved in methanol, filtered through a 0.22 μm organic filtration and analyzed by HPLC. The K value can be calculated as K = Aupper/Alower (Table I), where Aupper and Alower were the HPLC peak areas of target compounds in the upper and lower phase, respectively. And the separation factor (α) was defined as the ratio of partition coefficients (K) between two adjacent compounds by the equation α = K1/K2 (K1 ⩾ K2). Preparation of the two-phase solvent systems and the sample solution Each solvent according to the selected biphasic systems was added to a 1000 mL separatory funnel, shaken vigorously and equilibrated at room temperature. The biphasic systems of each phase were separated and ultrasonically degassed for 30 min before used. The upper phase was used as the stationary phase and lower phase as the mobile phase. The sample solutions for HSCCC were prepared by dissolving the sample enriched with D101 resin in 20 mL lower phase to an appropriate concentration. HSCCC separation procedure The upper organic phase was pumped into the multiplayer coiled column as stationary phase with no rotation. Then the apparatus was rotated at 900 rpm, while the lower phase was pumped through the inlet of column at a flow rate of 2 mL/min. After a hydrodynamic equilibrium was reached, the lower phase was started to elute from the outlet of column. Then, the sample solution was injected through the sample port. The separated sample components flowed out of the column outlet and were continuously monitored at 325 nm. Each peak fraction was collected manually according to the chromatograph and then evaporated under vacuum at 42°C for further HPLC analysis. After collecting all the components of the mobile phase, rotation was stopped and the column was flushed with nitrogen. HPLC detection The caffeoylquinic acids were analyzed by Agilent 1200 liquid chromatography system equipped with a Luna C18 column (250 × 4.6 mm i.d.; 5 μm; Phenomenex, USA), and the wavelength was set at 325 nm. The elution solvents were composed of mobile phase A (0.5% phosphoric acid aqueous solution) and mobile phase B (acetonitrile). The gradient elution conditions were as follows: 0–25 min and 10–13% B. The total run time was 25 min and the flow rate was constant at 1 mL/min. The injection volume of all samples was 20 μL, and the column temperature was maintained at 25°C. UHPLC-DAD-ESI-MS/MS and NMR analysis The chemical structure identification of three target compounds was performed by MS, 1H NMR and 13C NMR. ESI-MS/MS spectra were obtained on Agilent 6460 Accurate-Mass (Agilent Technologies, Santa Clara, CA) in negative mode. NMR spectra were taken on Bruker 400 MHz spectrometer (Bruker Bio Spin Corporation, Billerica, MA). Figure 2 Open in new tabDownload slide Effects of different pH and number of sequential extractions on extraction rate of three caffeoylquinic acids in mulberry leaves. 1–5 represents the number of sequential extractions: 1, first time; 2, second time; 3, third time; 4, fourth time; 5, fifth time. Figure 2 Open in new tabDownload slide Effects of different pH and number of sequential extractions on extraction rate of three caffeoylquinic acids in mulberry leaves. 1–5 represents the number of sequential extractions: 1, first time; 2, second time; 3, third time; 4, fourth time; 5, fifth time. Figure 3 Open in new tabDownload slide Selection and optimization of macroporous resin. (A) Adsorption capacities and desorption ratios. (B) Dynamic breakthrough curves. (C) Dynamic desorption curves. (D) Cleaning volume. Figure 3 Open in new tabDownload slide Selection and optimization of macroporous resin. (A) Adsorption capacities and desorption ratios. (B) Dynamic breakthrough curves. (C) Dynamic desorption curves. (D) Cleaning volume. Results Enrichment of three caffeoylquinic acid isomers by D101 A D101 resin was selected to enrich three caffeoylquinic acids before HSCCC separation due to the adsorption capacities and desorption ratios for caffeoylquinic acids. The optimal enrichment conditions for target compounds were feed volume 8/3 BV, a gradient elution distilled water 1 BV and 10% ethanol 8 BV. HSCCC separation HSCCC separation with the solvent system, ethyl acetate–water (1:1, v/v), was performed at a flow rate of 2 mL/min with the rotational speed of 800 rpm in 150 min. Under the optimized conditions, three caffeoylquinic acids were isolated and purified. The HSCCC chromatogram was shown in Figure 1A. The HPLC analysis of the sample enriched by D101 resin and the target compounds by preparative HSCCC were shown in Figure 1B. The structural identification of three target compounds The structure of three target compounds was confirmed by ESI-MS/MS，1H NMR and 13C NMR (shown in Supplementary data Figures S1 and S2). The isolated compounds 1, 2 and 3 were identified as 3-CQA, 5-CQA and 4-CQA according to the literatures (31, 32). Compound 1 (peak A in Figure 3) was obtained with a white powder (methanol-d4). The negative ESI-MS spectrum (shown in Supplementary data Figure S1A): ESI-MS m/z 352.9 (M-H)−，179.0 (M-C7H10O5)− and 135.0 (M-C7H10O5-CO2)−. 1H NMR spectrum (400 MHz; methanol-d4; shown in Supplementary data Figure S2A) δ: 7.58 (1H, d, J = 15.9 Hz, H-7′), 7.04 (1H, d, J = 2.1 Hz, H-2′), 6.93 (1H，dd, J = 8.2, 2.1 Hz, H-6′), 6.75 (1H, d, J = 8.2 Hz, H-5′), 6.30 (1H, d, J = 15.9 Hz, H-8′), 5.35 (1H, q, J = 4.0 Hz, H-3), 4.14 (1H, td, J = 9.1, 4.2 Hz, H-5), 3.72 (1H, m, H-4), 1.95–2.15 (4H, m, H-2,6); 13C NMR (101 MHz; methanol-d4; shown in Supplementary data Figure S2A) δ: 177.10 (C-7), 167.61 (C-9′), 148.01 (C-4′), 145.39 (C-3′，7′), 126.57 (C-1′), 121.46 (C-6′), 115.06 (C-5′), 114.44 (C-8′), 113.71 (C-2′), 74.04 (C-1), 73.33 (C4), 71.58 (C-3), 66.97 (C-5), 40.02 (C-6), 35.35 (C-2). Table II Amount, purity and recovery of the target compounds Compounds name Loading amount (mg) The content of the target compounds in sample (mg) Amount obtained after HSCCC (mg) Purity of obtained fractiona (%) Recovery rateb (%) 3-CQA 50 2.39 1.47 98.99 60.81 100 4.77 3.78 96.97 76.87 200 9.55 8.53 94.13 84.12 400 19.10 16.42 93.75 80.59 500 23.87 20.88 90.23 78.93 600 28.64 25.14 87.27 76.60 5-CQA 50 9.47 7.11 98.45 73.88 100 18.95 18.06 98.31 93.69 200 37.90 38.35 97.45 98.61 400 75.80 80.61 93.62 99.56 500 94.75 99.49 90.45 94.97 600 113.70 95.83 64.02 53.96 4-CQA 50 4.78 2.79 98.75 57.64 100 9.56 8.05 98.63 82.99 200 19.13 15.61 97.89 79.87 400 38.26 37.85 95.22 94.21 500 47.82 46.65 90.44 88.23 600 57.39 60.81 32.00 33.91 Compounds name Loading amount (mg) The content of the target compounds in sample (mg) Amount obtained after HSCCC (mg) Purity of obtained fractiona (%) Recovery rateb (%) 3-CQA 50 2.39 1.47 98.99 60.81 100 4.77 3.78 96.97 76.87 200 9.55 8.53 94.13 84.12 400 19.10 16.42 93.75 80.59 500 23.87 20.88 90.23 78.93 600 28.64 25.14 87.27 76.60 5-CQA 50 9.47 7.11 98.45 73.88 100 18.95 18.06 98.31 93.69 200 37.90 38.35 97.45 98.61 400 75.80 80.61 93.62 99.56 500 94.75 99.49 90.45 94.97 600 113.70 95.83 64.02 53.96 4-CQA 50 4.78 2.79 98.75 57.64 100 9.56 8.05 98.63 82.99 200 19.13 15.61 97.89 79.87 400 38.26 37.85 95.22 94.21 500 47.82 46.65 90.44 88.23 600 57.39 60.81 32.00 33.91 aThe purities of the obtained fractions from HSCCC determined by HPLC. b|$\mathrm{Recovery}\ \mathrm{rate}=\frac{\Big(\mathrm{amount}\ \mathrm{obtained}\ \mathrm{fraction}\ \mathrm{from}\ \mathrm{HSCCC}\Big)\times \Big(\mathrm{purity}\ \mathrm{of}\ \mathrm{the}\ \mathrm{obatained}\ \mathrm{fraction}\Big)}{\Big(\mathrm{the}\ \mathrm{content}\ \mathrm{of}\ \mathrm{the}\ \mathrm{target}\ \mathrm{compouds}\ \mathrm{in}\ \mathrm{sample}\Big)\times \Big(\mathrm{loading}\ \mathrm{amount}\Big)}\times 100$| Open in new tab Table II Amount, purity and recovery of the target compounds Compounds name Loading amount (mg) The content of the target compounds in sample (mg) Amount obtained after HSCCC (mg) Purity of obtained fractiona (%) Recovery rateb (%) 3-CQA 50 2.39 1.47 98.99 60.81 100 4.77 3.78 96.97 76.87 200 9.55 8.53 94.13 84.12 400 19.10 16.42 93.75 80.59 500 23.87 20.88 90.23 78.93 600 28.64 25.14 87.27 76.60 5-CQA 50 9.47 7.11 98.45 73.88 100 18.95 18.06 98.31 93.69 200 37.90 38.35 97.45 98.61 400 75.80 80.61 93.62 99.56 500 94.75 99.49 90.45 94.97 600 113.70 95.83 64.02 53.96 4-CQA 50 4.78 2.79 98.75 57.64 100 9.56 8.05 98.63 82.99 200 19.13 15.61 97.89 79.87 400 38.26 37.85 95.22 94.21 500 47.82 46.65 90.44 88.23 600 57.39 60.81 32.00 33.91 Compounds name Loading amount (mg) The content of the target compounds in sample (mg) Amount obtained after HSCCC (mg) Purity of obtained fractiona (%) Recovery rateb (%) 3-CQA 50 2.39 1.47 98.99 60.81 100 4.77 3.78 96.97 76.87 200 9.55 8.53 94.13 84.12 400 19.10 16.42 93.75 80.59 500 23.87 20.88 90.23 78.93 600 28.64 25.14 87.27 76.60 5-CQA 50 9.47 7.11 98.45 73.88 100 18.95 18.06 98.31 93.69 200 37.90 38.35 97.45 98.61 400 75.80 80.61 93.62 99.56 500 94.75 99.49 90.45 94.97 600 113.70 95.83 64.02 53.96 4-CQA 50 4.78 2.79 98.75 57.64 100 9.56 8.05 98.63 82.99 200 19.13 15.61 97.89 79.87 400 38.26 37.85 95.22 94.21 500 47.82 46.65 90.44 88.23 600 57.39 60.81 32.00 33.91 aThe purities of the obtained fractions from HSCCC determined by HPLC. b|$\mathrm{Recovery}\ \mathrm{rate}=\frac{\Big(\mathrm{amount}\ \mathrm{obtained}\ \mathrm{fraction}\ \mathrm{from}\ \mathrm{HSCCC}\Big)\times \Big(\mathrm{purity}\ \mathrm{of}\ \mathrm{the}\ \mathrm{obatained}\ \mathrm{fraction}\Big)}{\Big(\mathrm{the}\ \mathrm{content}\ \mathrm{of}\ \mathrm{the}\ \mathrm{target}\ \mathrm{compouds}\ \mathrm{in}\ \mathrm{sample}\Big)\times \Big(\mathrm{loading}\ \mathrm{amount}\Big)}\times 100$| Open in new tab Compound 2 (peak C in Figure 3) was obtained with a white powder (methanol-d4). The negative ESI-MS spectrum (shown in Supplementary data Figure S1B): ESI-MS m/z 352.9 (M-H)−, 191.0 (M-C9H6O3)−; 1H NMR (400 MHz; methanol-d4; shown in Supplementary data Figure S2B) δ: 7.56 (1H, d, J = 15.9 Hz, H-7′), 7.05 (1H, d, J = 2.1 Hz, H-2′), 6.95 (1H, dd, J = 8.2, 2.1 Hz, H-6′), 6.78 (1H, d, J = 8.2 Hz, H-5′), 6.26 (1H, d, J = 15.9 Hz, H-8′), 5.33 (1H, m, H-3), 4.15 (1H, ddt, J = 10.7, 6.3, 3.2 Hz, H-5), 3.72 (1H, m, H-4), 2.06–2.21 (4H, m, H-2,6); 13C NMR (101 MHz; methanol-d4; shown in Supplementary data Figure S2B) δ: 175.63 (C-7), 167.25 (C-9), 148.15 (C-4′), 145.65 (C-3′), 145.38 (C-7′), 126.40 (C-1′), 121.55 (C-6′), 115.07 (C-5′), 113.87 (C-8′), 113.79 (C-2′), 74.77 (C-1), 72.10 (C-4), 70.58 (C-5), 69.92 (C-3), 37.41 (C-2), 36.83 (C-6). Compound 3 (peak B in Supplementary data Figure 3) was obtained with a white powder (methanol-d4). The negative ESI-MS spectrum (shown in Supplementary data Figure S1C): ESI-MS m/z 352.9 (M-H)−, 191.0 (M- C9H6O3)−, 173.0 (M-C7H10O5-H2O)−. 1H NMR (400 MHz; methanol-d4, shown in Supplementary data Figure S2C) δ: 7.65 (1H, d, J = 15.9 Hz, H-7′), 7.08 (1H, m, H-2′), 6.96 (1H, dd, J = 8.2, 2.0 Hz, H-6′), 6.77 (1H, m, H-5′), 6.37 (1H, d, J = 15.8 Hz, H-8′), 4.81 (1H, m, H-5), 4.30 (1H, s, H-3), 3.60(1H, q, H-4), 2.17 (2H, s, H-6), 1.98 (2H, m, H-2); 13C NMR (101 MHz; methanol-d4; shown in Supplementary data Figure S2C) δ: 176.62 (C-7), 167.58 (C-9′), 148.14 (C-4′), 145.69 (C-3′), 143.93 (C-7′), 126.48 (C-1′), 121.54 (C-6′), 115.08 (C-5′), 114.00 (C-8′), 113.76 (C-2′), 77.77 (C-1), 75.05 (C-5), 68.14 (C-4), 64.40 (C-3), 41.11 (C-2), 37.27 (C-6). Discussion Selection and optimization of macroporous resin Caffeoylquinic acids are polyhydroxy phenolic acids. Under acidic conditions, caffeoylquinic acids mostly exist in neutral state, and hydrophobicity is enhanced, which is more conducive to enter organic phase. Therefore, the organic acid molecules can be transferred to organic phase as much as possible by adjusting the pH of the sample solution. The effects of different pH and extraction times on extraction rate of three caffeoylquinic acids were showed in Supplementary data Figure S3 and Figure 2. The results showed that the extraction rate of target compounds was highest when pH = 4. As can be seen from Figure 2, the same extraction rate could be reached after twice extraction when pH = 4, while five times extraction when pH = 4. In order to select the most suitable resin for adsorption and separation of target compounds, adsorption and desorption capacities on six commercial resins were shown in Figure 3A. As seen from Figure 3A, compared with other resins, D101 had a stronger adsorption capacity for the target compounds and was easier to elute. Several additional macroporous resin optimization parameters were also examined, i.e., feed volume, elution system and cleaning volume (Figures 3B–D). The results indicated that the optimal enrichment conditions for target compounds were feed volume 8/3 BV, a gradient elution distilled water 1 BV and 10% ethanol 8 BV. Under these optimal parameters, preparative scale of the caffeoylquinic acids by D101 resin was carried out, and 5.86 g enriched sample was obtained. After one-step enrichment, the contents of 3-CQA, 5-CQA and 4-CQA in enriched sample reached 4.77%, 18.95% and 9.84%, which were 3.13-fold, 4.57-fold and 4.78-fold to those in n-butanol extract, respectively. Hence, the proposed method can effectively separate these three caffeoylquinic acids from mulberry leaves. Selection of two-phase solvent system Generally, a successful HSCCC separation mainly depends on the selection of appropriate biphasic solvent systems. Target compounds with smaller K value can be eluted closer to the solvent front with lower resolution, while compound with a larger K value provide a better resolution but it can cause broader, more dilute peaks due to a longer elution time (33). Therefore, to get a satisfactory separation and short settling (33, 34), an ideal K value of the target compounds should be between 0.2 and 2. Then, the separation factor (α) should be >1.5 to obtain an effective separation. Due to the high polarity of the target compounds, a series of polar solvent systems were examined in the present work. Table I showed the values of K and α of target compounds in different biphasic solvent systems composed of ethyl acetate-n–butanol–acetic acid–water. Among the solvent systems, the values of K and α of ethyl acetate–water (1:1, v/v) was found to be satisfactory for separating three caffeoylquinic acids. Optimization of sample loading amount It is generally true that the more increasement of loading sample, the more separation of target compounds can be obtained by HSCCC. However, it is often limited by the volume of the spiral tube. Different amounts of the sample enriched by D101 resin were dissolved into lower phase and injected through the sample port. The amount, purity and recovery of the target compounds with different sample loading amounts were shown in Table II and Supplementary data Figure S4. As we can see from Table II, the purity and recovery rate of three caffeoylquinic acids were both >90% in the sample loading amounts from 50 to 500 mg. However, as the amount of the sample was increased to 600 mg, the separation and preparation efficiency of the target compounds decreased sharply, resulting in poorer purity and lower recovery. It can be seen from the Supplementary data Figure S4 that 5-CQA and 4-CQA were not separated completely. And when the amount of the sample increased from 400 to 500 mg, its purity and recovery decreased, and the final yield failed increasing much. In a comprehensive consideration, the sample loading amount of 400 mg was selected to separate the caffeoylquinic acids while the purity, recovery and final yield reached higher values. Conclusions In the present study, a strategy based on liquid–liquid extraction with acid condition and HSCCC could provide highly efficient preparative separation of three caffeoylquinic acid isomers from mulberry leaves. By adjusting pH value of the solution, the caffeoylquinic acids were efficiently and quickly extracted into the organic phase, which saved much time and reagents, and reduced the industrial costs. The present study provided a feasible, economical and efficient method for preparative separation of natural products from herbal materials. Moreover, it could be easy to scale-up for food and pharmaceutical industrial application. Funding The authors gratefully acknowledge the financial supports by Fundamental Research Funds for the Central Universities (2018ZY22, BLX201805 and 2572017AA08); Key R&D Program of Ministry of Science and Technology—Regulation of Key Characters of Mulberry (2018YFD1000602); Wild Plant Protection and Management Project (2018HXFWLXY028); Forestry Industry Standard Revision Project (2016-LY-050); and National Natural Science Fund (31800509). References 1. 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TI - An Efficient Strategy Based on Liquid–Liquid Extraction With Acid Condition and HSCCC for Rapid Enrichment and Preparative Separation of Three Caffeoylquinic Acid Isomers From Mulberry Leaves
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz050
DA - 2019-08-16
UR - https://www.deepdyve.com/lp/oxford-university-press/an-efficient-strategy-based-on-liquid-liquid-extraction-with-acid-VPVXMXW0Fj
SP - 738
VL - 57
IS - 8
DP - DeepDyve
ER -