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Simple and efficient preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography
Separation and Purification Technology 120 (2013) 310–318
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Simple and efficient preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography Fei-Yue Ma, Meng Luo, Chun-Jian Zhao, Chun-Ying Li, Wei Wang, Cheng-Bo Gu, Zuo-Fu Wei, Yuan-Gang Zu, Yu-Jie Fu ⇑ State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 3 August 2013 Accepted 29 September 2013 Available online 9 October 2013 Keywords: Flash chromatography Macroporous resin Separation Dalbergia odorifera T. Chen leaves
a b s t r a c t In this study, a simple rand efficient protocol is developed for preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography. The adsorption and desorption capacity of 14 macroporous adsorption resins for biochanin A and genistein were evaluated, and AL-2 resin showed better properties for biochanin A and genistein. After treatment with AL-2 resin, the contents of biochanin A and genistein in the enriched product were 6.60-fold and 6.41-fold increased with recovery yields of 87.13% and 84.60%, respectively. Furthermore, the operating parameters of flash chromatography were optimized. The optimal conditions were as follows: stationary phase: silica gel, elution system: n-hexane/ethyl acetate, sample/silica gel ratio: 1.3:40 and flow rate: 50 mL/min. After one flash chromatography run, purities of biochanin A and genistein effectively reached over 95%, their recovery yields were 80.13% and 73.11%, respectively. The developed protocol was simple, efficient, scalable and economical, which represented an excellent alternative for the separation and purification of bioactive compounds from plants. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids, a large category of plant polyphenol secondary metabolites, are widely distributed in medicinal herbs, fruits, teas, etc. [1]. They possessed particular interest with regard to human health effects [2]. As a kind of flavonoids, isoflavonoids can be potentially used as clinical therapeutic agents, food additive and health care products [3–8]. D. odorifera leaves, a natural renewable resource, usually were discarded as useless material. Our previous studies found that biochanin A and genistein are the main active constituents present in D. odorifera leaves [9]. Biochanin A and genistein are naturally occurring plant-derived phytoestrogen, and possess anticancer, antioxidant and antiosteoporosis effect. In view of these beneficial effects, it is necessary to obtain high purity of biochanin A and genistein for further medical studies and applications. Preparation of isoflavonoids from plant extract is a challenging task because plant extract was a multi-component system. Over
⇑ Corresponding author at: State Engineering Laboratory for Bio-Resource EcoUtilization, Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86 451 82190535. E-mail address: [email protected] (Y.-J. Fu). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.09.035
the past decade, various physical and chemical techniques for the enrichment of bioactive compounds from plant extract have been investigated, such as liquid–liquid extraction [10,11], membrane filtration [12], ion exchange [13], solid phase microextraction [14] and adsorption–desorption [15]. Among them, adsorption– desorption is considered as an economical and efficient method for preparative enrichment of bioactive components from plant extracts. Macroporous resins adsorption technology has been increasingly viewed as a representative adsorption–desorption for enriching bioactive components [16–18]. Macroporous resins have unique superiorities, such as high mechanical strength, high selectivity, good acid and alkali resistance, low cost, convenience and easy regeneration [19,20]. Open column chromatography is traditionally used for separating bioactive components from plant extracts. However, this method is time-consuming, laborious and requires large volumes of solvents [1]. These limitations warranted to explore a fast and efficient method for preparation of natural products from plant extract. Flash chromatography, also known as medium pressure chromatography, can efficiently overcome these limitations. It is considered as a fast, inexpensive and efficient separation technique for the separation of natural active components from extracts, and is easy to handle. This technique is an excellent alternative for slow and often inefficient gravityfed chromatography. Compared to traditional gravity-fed
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chromatography, flash chromatography gives a greatly improved reproducibility, higher resolution and recovery yield, and a reduced consumption of organic solvents. Moreover, with a UV detector and an automatic fraction collector, the sample solvent could be collected according to chromatograms profile during elution process. The aim of this work was to establish a simple and efficient protocol for preparation of biochanin A and genistein from D. odorifera leaves using macroporous resin followed by flash chromatography. Up to now, there has never been the report on separation of biochanin A and genistein by flash chromatography. The operating parameters of macroporous resin and flash chromatography were optimized. Hopefully, this work is helpful for the scale-up application for the production of biochanin A and genistein from the D. odorifera leaves and other plants.
2. Materials and methods 2.1. Materials, chemicals and adsorbents The Dalbergia odorifera T. Chen leaves were collected from Hainan Province China, and authenticated by Professor Shaoquan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, China. The leaves were dried in shade at room temperature, powdered by a disintegrator and then stored in dark. Reference compounds biochainin A (40 ,5,7-trihydroxyisoflavone-7-glucoside,P98%) and genistein (40 ,5,7-trihydroxyisoflavone,P98%) were purchased from Fluka (Buchs, Switzerland). Reagents of HPLC grade including methanol and formic acid were purchased from J&K Chemical LTD. (Beijing, China). Deionized water was produced by a Millipore Direct-Q purification system (Millipore Corp., Bedford, MA, USA). Ethanol of analytical grade was obtained from Tianjin Chemical Reagents Co. (Tianjin, China). The solvents (n-hexane, ethyl acetate, chloroform and petroleum) used for preparative flash chromatography were of analytical grade. Fourteen macroporous resins including AB-8, ADS-5, ADS-11, D101, FL-1, FL-2, FL-3, HPD500, HPD826, HPD-D, ME-2, AL-2, SA-3 and NKA-9 were purchased from Nankai Hecheng S&T (Tianjin, China) and Bonchem (Hebei, China). Their polarities ranged from non-polar to strong polar. All the resins were pretreated according to the manufacturers’ recommendation prior to use in order to remove the monomers and porogenic agents trapped inside the pores during the synthesis process [4,21]. The moisture contents of the tested resins were determined by drying the beads at 100 °C to constant weight in a drying oven for over 24 h. Toyopearl HW-40S gel resin and silica gel (300–400 mesh) were purchased from Tosoh Corporation (Tokyo, Japan) and Qingdao Meigao Chemical Co. Ltd., (Qingdao, China), respectively. TLC (Kieselgel GF254) was purchased from Taizhou Luqiao Sijia Biochemical Plastic Factory (Taizhou, China).
2.3. HPLC analysis of biochanin A and genistein Biochanin A and genistein were analyzed using an Agilent 1200 HPLC system. Chromatographic separation was carried out on a HIQ Sil C18V reversed-phase column (250 mm 4.6 mm i.d., 5 lm). All samples were filtered through 0.45 lm nylon membranes prior to HPLC analysis. The mobile phase consisted of acetonitrile (A) and water–formic acid (B). The gradient elution program was as follows: 0–5 min, 5–10% A; 5–10 min, 10–28% A; 10– 30 min, 28–60% A; 30–35 min, 60% A; 35–37 min, 60–80% A; 37– 42 min, 80–100% A; 42–55 min, 100% A. The injection volume was 5 lL. The flow rate and column temperature was 1 mL/min and 30 °C, respectively. Biochanin A and genistein were quantified at wavelength 262 nm. The chromatographic peaks of the analytes were confirmed by comparing their retention times and UV spectrum with those of the reference compounds. Eight experimental points were employed for establishing a calibration curve. The regression lines for biochanin A and genistein were Y = 84 698 x + 78.7 (R2 = 0.9949) and Y = 71 148 x + 1.37 (R2 = 0.9927), where Y is the peak area of analyte, and x is the concentration of reference compound (mg/mL). 2.4. Enrichment of biochanin A and genistein by macroporous resin 2.4.1. Screening of macroporous resins Macroporous resins can selectively adsorb and desorb constituents from sample solutions due to their specific physical and chemical properties. The adsorption and desorption capacity of different resins towards biochanin A and genistein were investigated. The adsorption tests were performed as follows: pre-weighed hydrated resins (equal to about 1.0 g dry resin) and 100 mL sample solution were added into 250 mL flasks with stopper. The flasks were shaken in an incubation shaker (120 rpm) for 4 h at 25 °C. After adsorption, the solutions were separated from the resins and analyzed by HPLC. Then, the resins were subsequently desorbed with 100 mL 80% ethanol solution. The flasks were continually shaken (120 rpm) for 6 h at 25 °C. The contents of biochanin A and genistein in desorption solutions were determined by HPLC. The adsorption properties of resins were evaluated based on the adsorption and desorption capacities and ratio of desorption. The equations were as follows: Adsorption evaluation:
Qe ¼
Pulverized D. odorifera leaves (5 kg) were extracted with 30 L of 80% ethanol at room temperature for 3 days, repeated twice. The filtered solutions were gathered and concentrated to dryness by removing the ethanol solvent using a rotary evaporator device (RE52AA, Shanghai Huxi Instrument Co., China) at 40 °C. The dried extracts were obtained, and dissolved in 30% ethanol to get sample solution (5 mg extract/mL) at the concentration of 0.2058 mg/mL for biochanin A and 0.0530 mg/mL for genistein, respectively (Table 1).
ðC 0 C e ÞV i W
where Qe is the adsorption capacity at adsorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of solute in the solutions, respectively (mg/mL); Vi is the volume of sample solution, and W is the weight of the dry resin. Desorption evaluation:
D¼
Cd V d 100% ðC 0 C e ÞV i
Qd ¼ 2.2. Preparation of D. odorifera leaves extracts
311
CdV d W
where Qd is the desorption capacity after adsorption equilibrium (mg/g resin); D is the desorption ratio (%); Cd is the concentration of solute in the desorption solution (mg/mL); Vd is the volume of the desorption solution (mL); C0, Ce, W and Vi are the same as described above. 2.4.2. Determination of macroporous resins/sample ratio A series of adsorptions was carried out with different macroporous resins/sample ratios (2:1, 3:1, 4:1, 4.5:1 and 5:1, w/w) to determine the optimal macroporous resins/sample ratio.
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Table 1 The purities and yields of the compounds in proposed separation procedure. Compound
Procedure
Purity (%)
Yield (%)
Total yield (%)
Biochanin A
Crude extract Macroporous resin enrichment Separation by silica gel flash chromatography Crude extract Macroporous resin enrichment Separation by silica gel flash chromatography
4.12 27.20 >95
— 87.13 80.13
69.82
1.06 6.79 >95
— 84.60 73.11
61.85
Genistein
Pre-weighed resins were added to the sample solution. The mixture was shaken in an incubation shaker (120 rpm) for 4 h at 25 °C. After adsorption, the solutions were separated from the resins and analyzed by HPLC. The adsorption ratios of biochanin A and genistein were calculated according to the following equation:
E¼
C0 Ce 100% C0
where E is the adsorption ratio (%), which is the percent of the adsorbed quantity to the initial quantity under equilibrium; C0 and Ce are the initial and equilibrium concentrations of solute in the solutions, respectively (mg/mL). After determining the adsorption resin and resin/sample ratio based on the above experiments, scaled-up enrichment of
Fig. 1. The actual flash chromatography system (A), schematic representation of the flash chromatography system (B), and schematic diagram of purification process for biochanin A and genistein from D. odorifera leaves (C). a – mobile phase; b – pump; c – chromatographic column; d – UV detector; e –automatic collector; f – data acquisition system.
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Fig. 2. Adsorption capacities and desorption ratios of biochanin A (A) and genistein (B) on different macroporous resins.
biochanin A and genistein was carried out to obtain enriched product for flash chromatography experiment.
TLC, the eluate was divided into three fractions: fraction A (Fr.A) containing biochanin A; fraction B (Fr.B) containing biochanin A and genistein, and fraction C (Fr.C) containing genistein.
2.5. Separation and purification of biochanin A and genistein by flash chromatography The separation and purification of biochanin A and genistein were performed on a commercial flash chromatography system (Leisure, Shanghai, China) equipped with a binary liquid pump, a UV detector and an automatic collector. The actual flash chromatography system and schematic representation of the flash chromatography system were shown in Fig. 1A and B. The enriched product of biochanin A and genistein was dissolved in methanol. The same amount of silica gel (300– 400 mesh) was added to the sample solutions, and then removed solvent by rotary evaporator device at 40 °C. The mixture of enriched product and silica gel was loaded on a column (26 mm 125 mm I.D.) which was pre-packed with silica gel (300–400 mesh). Separation of biochanin A and genistein was achieved using gradient elution system. The eluate was fractionated and collected by automatic collector according to the chromatograms at 262 nm. Based on the chromatograms profile and
Fig. 3. Effect of macroporous resins/sample ratio on the adsorption ratios of biochanin A and genistein.
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Fig. 4. HPLC chromatograms of crude extract (A), enriched product with AL-2 resin (B) and purified biochanin A (C) and genistein (D).
Then, the fraction A, B and C were evaporated by rotary evaporation, respectively. The fraction A and C were analyzed by HPLC to determine the purities and recovery yields of obtained biochanin A and genistein, respectively. In order to screen optimum elution system, three elution systems, n-hexane–ethyl acetate, n-hexane– chloroform, n-hexane–acetone, were used as gradient elution solvent. The optimum sample/silica gel ratio and flow rate were also studied systematically in this work. Another chromatogram column (26 mm 125 mm I.D.) was packed with Toyopeal HW-40S gel resin. The enriched product of biochanin A and genistein (1.0 g) was dissolved in 6 mL methanol, and then loaded on the Toyopeal HW-40 gel resin column. The biochanin A and genistein were eluted with pure methanol. The flow rate was kept at 30 mL/min. The schematic diagram of purification process for biochanin A and genistein from D. odorifera leaves was shown in Fig. 1C.
3. Results and discussion 3.1. Enrichment by macroporous resin 3.1.1. Screening of macroporous resins To enrich effectively biochanin A and genistein from D. odorifera leaves extracts by macroporous resins, the optimum type of macroporous resin was screened firstly. The properties of these resins were compared in terms of their adsorption and desorption capacities as well as desorption ratios. As can be seen from Fig. 2, the adsorption capacities of FL-1, HPD-500 and SA-3 resins towards genistein were higher, and the adsorption capacities of FL-1, ME2 and HPD-500 resins towards biochanin A were better. The adsorption capacity not only correlates with the physical and chemical properties of adsorbent, but also with the size and chemical features of the adsorbed substance. However, their lower
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desorption capacities resulted in a lower desorption ratio. Desorption ratios of AL-2 resin for genistein and biochanin A were obviously higher than those of others, and AL-2 resin possessed moderate adsorption capacity towards genistein and biochanin A. Therefore, AL-2 resin was chosen as the optimum resin for the enrichment. 3.1.2. Effect of macroporous resin /sample ratio on adsorption ratio The effect of macroporous resins/sample ratio on the adsorption ratio was investigated at macroporous resins/sample ratio of 2:1– 5:1. The results are shown in Fig. 3. It can be seen that the adsorption ratios increased with the macroporous resins/sample ratio. When the macroporous resins/sample ratio is 3:1, the adsorption ratio of bochanin A reached 90%. But in this case, the adsorption ratio of genistein was till lower. When the macroporous resins/sample ratio is 5:1, the adsorption ratios of bochanin A and genistein reached 97.45% and 90.24%. Considering simultaneous enrichment of biochanin A and genistein, a macroporous resins/sample ratio of 5:1 was chosen. In order to obtain enriched product for the following flash chromatography experiment, scaled-up enrichment of biochanin A and genistein was carried out. The chromatograms of the samples before and after treatment with AL-2 resin were shown in Fig. 4A and B. The contents of biochanin A and genistein in crude extracts were 4.12% and 1.06%. After the enrichment on AL-2 resin, the contents of biochanin A and genistein in the enriched product reached 27.20% and 6.79%, which were 6.60-fold and 6.41-fold to those in crude extract, respectively, and the recovery yields were 87.13% and 84.60%, respectively (Table 1). This process achieved easy and effective enrichment of biochanin A and genistein by using AL-2 resin.
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resin which is based on hydroxylated methacrylic polymer [22]. Recently, due to high mechanical strength, good chemical stability and much better regeneration efficiency, it has been more and more widely applied in the separation and purification of natural products [22–25]. Especially, it exhibited potential capacity for separation and purification of flavonoids [22,23]. Fig. 5A showed the chromatogram profiles of separating biochanin A and genistein by silica gel flash chromatography. Separation of biochanin A and genistein was achieved by the following elution program: 0– 13.5 min, n-hexane–ethyl acetate (10:1, v/v); 13.5–45 min, n-hexane–ethyl acetate (6:1, v/v). The flow rate was kept at 30 mL/min. The sample/silica gel ratio was 1:40 (1.0 g enrichment product: 40 g silica gel). Based on the chromatograms profile and TLC, we collected about 21–28 min and 32.5–38 min as Fr. A and C to yield biochanin A and genistein, respectively. After HPLC analysis, the
3.2. Separation and purification of biochanin A and genistein by flash chromatography 3.2.1. Selection of stationary phase Stationary phase played a decisive role in separation of biochanin A and genistein. Two stationary phases, silica gel and Toyopeal HW-40S, were tested in this experiment. Toyopeal HW-40S gel resin, as one of the novel stationary phase, is a molecular sieve gel
Fig. 5. Chromatogram profiles of enriched product by silica gel flash chromatography at 30 mL/min (A), Toyopeal HW-40S gel resin flash chromatography at 30 mL/ min (B).
Fig. 6. Effects of elution system (A), sample/silica gel ratio (B) and flow rate (C) on the yields of biochanin A and genistein.
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were separated by using n-hexane/ethyl acetate (6:1, v/v). Hence, n-hexane–ethyl acetate was used in silica gel flash chromatography for the separation and purification of biochanin A and genistein.
Fig. 7. Chromatogram profile of enriched product by silica gel flash chromatography under optimized conditions.
purities of biochanin A and genistein were more than 95%, the recovery yields reached 76.02% and 70.52%, respectively. Biochanin A and genistein with weak-polarity was hardly eluted on Toyopeal HW-40S column by aqueous methanol. So, pure methanol was employed as mobile phase. The flow rate was kept at 30 mL/min. As shown in Fig. 5B, biochanin A and genistein were not effectively separated. Only small amounts of Fr.A and C were obtained. Although the purities of purified biochanin A and genistein were more than 95%, their recovery yields were less than 15%. Hence, the silica gel flash chromatography was suitable for preparation of biochanin A and genistein from D. odorifera T. Chen leaves. The operating parameters of silica gel flash chromatography were optimized in the following experiments. 3.2.2. Effects of elution system Biochanin A and genistein are weak-polar compounds, so low polar solvents were required to separate on normal phase silica gel column by flash chromatography. Three elution systems with weak polarity (n-hexane–ethyl acetate, n-hexane–chloroform, nhexane–acetone) were tested. The results were shown in Fig. 6A. It was found only a little Fr.A and C were obtained using n-hexane–chloroform (CHCl3) and n-hexane–acetone systems because biochanin A and genistein could not be completely separated. Although the content of biochanin A and geinstein in Fr.A and Fr.C reached more than 90%, the recovery yields of biochanin A and genistein were extremely low (less than 45%). For n-hexane– ethyl acetate system, weak-polar impurities were effectively removed when n-hexane–ethyl acetate (10:1, v/v) elution system was employed. Biochanin A and genistein with similar polarity
3.2.3. Effects of sample/silica gel ratio High sample load may distort peak shape and cause an overall decrease in separation efficiency due to column overload. So, the influence of sample load on separation efficiency was also investigated by varying the sample/silica gel ratio from 4:40 to 1:40. The elution program was the same as Section 3.2.1. The results were shown in Fig. 6B. The yields of biochanin A and genistein increased drastically with decreasing sample/silica gel ratio from 4:40 to 2:40, but did not continue to significantly increase when the sample/silica gel ratio was below 1.3:40. Although the purity of biochanin A was more than 95% at the high sample/silica gel ratio (4:40, 2:40), the recovery yield of biochanin A was remarkably low. The same trend appeared in the recovery yield of genistein. As expected, when the sample/silica gel ratio decreased, separation efficiency gradually increased due to less sample load. So, satisfactory separation of biochanin A and genistein could be achieved with a sample/silica gel ratio of 1.3:40. 3.2.4. Effects of flow rate Flow rate, as a parameter of flash chromatography, effects the retention times, system pressure and separation efficiency. In order to keep the same volume of elution solvent, elution time was adjusted with different flow rate. Fig. 6C described the effect of flow rate on separation of biochanin A and genistein by silica gel flash chromatography. As shown in Fig. 6C, the recovery yields of biochanin A and genistein gradually increased with increasing flow rate from 30 to 50 mL/min. When the flow rate is lower, the time of contact between stationary phase and target compounds is longer, leading to difficulty in elution of target compounds from the chromatography column. When flow rate was increased to 70 mL/min, the recovery yields of biochanin A and genistein decreased apparently. The separation efficiency of chromatogram column is related to its plate number. Plate number rises firstly and then declines with increasing the flow rate. In a word, excessively high flow rate has negative effect on column chromatography. Moreover, higher flow rate resulted in increase of backpressure, which might require special facilities in industrial production, leading to increase of
Fig. 8. Negative ESI–MS and MS/MS spectra of purified biochanin A (A and B) and genistein (C and D) from D. odorifera leaves, respectively.
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instrument and consumable costs [26]. These factors become a greater concern in large-scale produce. So, a flow rate of 50 mL/ min was considered as optimal one. The corresponding gradient program was: 0–8 min, n-hexane–ethyl acetate (10:1, v/v); 8– 35 min, n-hexane–ethyl acetate (6:1, v/v). 3.2.5. Validation of Fash chromatography The enriched product of biochanin A and genistein (1.3 g) was dissolved in methanol. The same amount of silica gel (300– 400 mesh) was added to the sample solutions, and then removed solvent by rotary evaporator device at 40 °C. The mixture of enriched product and silica gel was loaded on a pre-packed 40 g silica gel (300–400 mesh) column (26 mm 125 mm I.D.). Separation of biochanin A and genistein was achieved using n-hexane and ethyl acetate as gradient elution solvent. The gradient program was: 0– 8 min, n-hexane–ethyl acetate (10:1, v/v); 8–35 min, n-hexane– ethyl acetate (6:1, v/v). The flow rate was kept at 50 mL/min. The chromatogram profile of enriched product by flash chromatography was shown in Fig. 7. Under the optimized conditions, 282.98 mg biochanin A and 64.52 mg genistein were obtained by one flash chromatography run. The purities of biochanin A and genistein both reached more than 95%, and their recovery yields were 80.13% and 73.11%, respectively (Table 1). HPLC chromatograms of purified biochanin A and genistein were shown in Fig. 4C and D, respectively. The proposed silica gel flash chromatography boasts high efficiency, low running costs and process automation, and it is a promising method for efficient preparation of biochanin A and genistein from D. odorifera T. Chen leaves. 3.3. Confirmation of biochanin A and genistein The structures of purified compounds were confirmed by MS spectra. ESI–MS spectra of purified biochanin A and genistein in Fig. 8A and C showed a molecular ion [M–H] at m/z 282.9 and 269.0, respectively. The MS/MS spectra of purified biochanin A and genistein in Fig. 8B and D were also consistent with those from previous publications [27]. 4. Conclusions In this work, AL-2 was used for effective enrichment of biochanin A and genistein from extract of D. odorifera leaves. After treatment with AL-2 resin, the contents of biochanin A and genistein in the enriched product reached 27.20% and 6.79% with recovery yields of 87.13% and 84.60%, respectively. Under the optimal conditions of flash chromatography, biochanin A and genistein were successfully separated after one flash chromatography run, and their purities were over 95% with the recovery yields of 80.13% and 73.11%. The total recovery yields of biochanin A and genistein reached 69.82% and 61.85%, respectively. These results indicated that the enrichment of macroporous resin followed by the separation of flash chromatography is an effective procedure for preparing high-purity biochanin A and genistein with advantages of high recovery yield, high efficiency, low running costs and process automation. This work provides a promising alternative for large-scale preparation of flavonoids from D. odorifera leaves or other plants. Acknowledgements The authors gratefully acknowledge the financial supports by Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Importation of International Advanced Forestry Science and Technology, National
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Forestry Bureau (2012-4-06) and Heilongjiang Province Science Foundation for Excellent Youths (JC200704). References [1] R.M. Uckoo, G.K. Jayaprakasha, B.S. Patil, Rapid separation method of polymethoxyflavones from citrus using flash chromatography, Sep. Purif. Technol. 81 (2011) 151–158. [2] Y. Zhang, J.J. Jiao, C.M. Liu, X.Q. Wu, Y. Zhang, Isolation and purification of four flavone C-glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography, Food Chem. 107 (2008) 1326–1336. [3] T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, a specific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem. 262 (1987) 5592–5595. [4] A. Rusin, Z. Krawczyk, G. Grynkiewicz, A. Gogler, J.Z. Puchalka, W. Szeja, Synthetic derivatives of genistein, their properties and possible applications, Acta Biochim. P. 57 (2010) 23–34. [5] C.E. Park, H. Yun, E.B. Lee, B.I. Min, H. Bae, W. Choe, I. Kang, S.S. Kim, J. Ha, The antioxidant effects of genistein are associated with AMP-activated protein kinase activation and PTEN induction in prostate cancer cells, J. Med. Food 13 (2010) 815–820. [6] Y. Zhang, W.F. Chen, W.P. Lai, M.S. Wong, Soy isoflavones and their bone protective effects, Inflammopharmacol 16 (2008) 213–215. [7] V. Sehdev, J.C.K. Lai, A. Bhushan, Biochanin a modulates cell viability invasion and growth promoting signaling pathways in HER-2-positive breast cancer cells, J. Oncol. (2009) 121458. [8] P. Mishra, A. Kar, R.K. Kale, Modulatory influence of prepubertal biochanin a exposure on mammary gland differentiation and expression of estrogen, receptor-a and apoptotic proteins, Phytother. Res. 23 (2009) 972–979. [9] D.Y. Zhang, Y.G. Zu, Y.J. Fu, M. Luo, C.B. Gu, W. Wang, X.H. Yao, Negative pressure cavitation extraction and antioxidant activity of biochanin A and genistein from the leaves of Dalbergia odorifera T Chen, Sep. Purif. Technol. 83 (2011) 91–99. [10] J.R. Kumar, H.I. Lee, J.Y. Lee, J.S. Kim, J.S. Sohn, Comparison of liquid–liquid extraction studies on platinum(IV) from acidic solutions using bis(2,4,4trimethylpentyl) monothiophosphinic acid, Sep. Purif. Technol. 63 (2008) 184– 190. [11] L.H. Wang, Y.H. Mei, F. Wang, X.S. Liu, Y. Chen, A novel and efficient method combining SFE and liquid–liquid extraction for separation of coumarins from Angelica dahurica, Sep. Purif. Technol. 77 (2011) 397–401. [12] P. Priyananda, V. Chen, Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration, Sep. Purif. Technol. 48 (2006) 113–120. [13] R.M.F. Espinosa, H.B.O. Oliveros, M.T. Olguín, M.R.P. Cueto, R.G.S. Vicente, Separation and treatment of ion-exchange resins used in cleaning systems of a research nuclear reactor, Chem. Eng. J. 188 (2012) 71–76. [14] G. Favaro, F. Magno, A. Boaretto, L. Bailoni, R. Mantovani, Traceability of asiago mountain cheese: a rapid, low-cost analytical procedure for its identification based on solid-phase microextraction, J. Dairy Sci. 88 (2005) 3426–3434. [15] Y.F. Liu, J.X. Liu, X.F. Chen, Y.W. Liu, D.L. Di, Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins, Food Chem. 123 (2010) 1027–1034. [16] M.R. Kim, W.C. Kim, D.Y. Lee, Recovery of narirutin by adsorption on a nonionic polar resin from a water-extract of Citrus unshiu peels, J. Food Eng. 78 (2007) 27–32. [17] A. Schieber, P. Hilt, H.U. Endress, A new process for the combined recovery of pectin and phenolic compounds from apple pomace, Innovative Food Sci. Emerg. Technol. 4 (2003) 99–107. [18] S. Liu, L.Z. Yi, Y.Z. Liang, Traditional Chinese medicine and separation science, J. Sep. Sci. 31 (2008) 2113–2137. [19] X.J. Li, Y.J. Fu, M. Luo, W. Wang, L. Zhang, C.J. Zhao, Y.G. Zu, Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography, J. Pharm. B. 61 (2012) 199–206. [20] Z.F. Wei, Y.G. Zu, Y.J. Fu, W. Wang, C.J. Zhao, M. Luo, T. Efferth, Resin adsorption as a means to enrich rare stilbenes and coumarin from pigeon pea leaves extracts, Chem. Eng. J. 172 (2011) 864–871. [21] B.Q. Fu, J. Liu, H. Li, L. Li, F.S.C. Lee, X.R. Wang, The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid, J. Chromatogr. A 1089 (2005) 18–24. [22] L.U. Hansen, M. Yanagishitag, Differential effect of brefeldin a on the biosynthesis of heparan sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture, J. Biol. Chem. 268 (1993) 17370–17376. [23] L. Liu, B.J. Xie, S.Q. Cao, E.N. Yang, X.Y. Xu, S.S. Guo, A-type procyanidins from Litchi chinensis pericarp with antioxidant activity, Food Chem. 105 (2007) 1446–1451. [24] S.Q. Cao, S.Y. Pan, X.L. Yao, H.F. Fu, Isolation and purification of anthocyanins from blood oranges by column chromatography, Agric. Sci. China 9 (2010) 207–215. [25] B. Hu, L. Wang, B. Zhou, X. Zhang, Y. Sun, H. Ye, L.Y. Hao, Q.H. Hu, G.X. Wang, X.X. Zeng, Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine
318
F.-Y. Ma et al. / Separation and Purification Technology 120 (2013) 310–318
alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection, J. Chromatogr. A 1216 (2009) 3223–3231. [26] S.H. Pyo, H.J. Choi, An improved high-performance liquid chromatography process for the large-scale production of paclitaxel, Sep. Purif. Technol. 763 (2011) 78–384.
[27] R. Vessecchi, G.J. Zocolo, D.R. Gouvea, F. Hübner, B. Cramer, M.R.R. de Marchi, H.U. Humpf, N.P. Lopes, Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies, Rapid Commun. Mass Spectrom. 25 (2011) 2020–2026.
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Simple and efficient preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography Fei-Yue Ma, Meng Luo, Chun-Jian Zhao, Chun-Ying Li, Wei Wang, Cheng-Bo Gu, Zuo-Fu Wei, Yuan-Gang Zu, Yu-Jie Fu ⇑ State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 3 August 2013 Accepted 29 September 2013 Available online 9 October 2013 Keywords: Flash chromatography Macroporous resin Separation Dalbergia odorifera T. Chen leaves
a b s t r a c t In this study, a simple rand efficient protocol is developed for preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography. The adsorption and desorption capacity of 14 macroporous adsorption resins for biochanin A and genistein were evaluated, and AL-2 resin showed better properties for biochanin A and genistein. After treatment with AL-2 resin, the contents of biochanin A and genistein in the enriched product were 6.60-fold and 6.41-fold increased with recovery yields of 87.13% and 84.60%, respectively. Furthermore, the operating parameters of flash chromatography were optimized. The optimal conditions were as follows: stationary phase: silica gel, elution system: n-hexane/ethyl acetate, sample/silica gel ratio: 1.3:40 and flow rate: 50 mL/min. After one flash chromatography run, purities of biochanin A and genistein effectively reached over 95%, their recovery yields were 80.13% and 73.11%, respectively. The developed protocol was simple, efficient, scalable and economical, which represented an excellent alternative for the separation and purification of bioactive compounds from plants. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids, a large category of plant polyphenol secondary metabolites, are widely distributed in medicinal herbs, fruits, teas, etc. [1]. They possessed particular interest with regard to human health effects [2]. As a kind of flavonoids, isoflavonoids can be potentially used as clinical therapeutic agents, food additive and health care products [3–8]. D. odorifera leaves, a natural renewable resource, usually were discarded as useless material. Our previous studies found that biochanin A and genistein are the main active constituents present in D. odorifera leaves [9]. Biochanin A and genistein are naturally occurring plant-derived phytoestrogen, and possess anticancer, antioxidant and antiosteoporosis effect. In view of these beneficial effects, it is necessary to obtain high purity of biochanin A and genistein for further medical studies and applications. Preparation of isoflavonoids from plant extract is a challenging task because plant extract was a multi-component system. Over
⇑ Corresponding author at: State Engineering Laboratory for Bio-Resource EcoUtilization, Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86 451 82190535. E-mail address: [email protected] (Y.-J. Fu). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.09.035
the past decade, various physical and chemical techniques for the enrichment of bioactive compounds from plant extract have been investigated, such as liquid–liquid extraction [10,11], membrane filtration [12], ion exchange [13], solid phase microextraction [14] and adsorption–desorption [15]. Among them, adsorption– desorption is considered as an economical and efficient method for preparative enrichment of bioactive components from plant extracts. Macroporous resins adsorption technology has been increasingly viewed as a representative adsorption–desorption for enriching bioactive components [16–18]. Macroporous resins have unique superiorities, such as high mechanical strength, high selectivity, good acid and alkali resistance, low cost, convenience and easy regeneration [19,20]. Open column chromatography is traditionally used for separating bioactive components from plant extracts. However, this method is time-consuming, laborious and requires large volumes of solvents [1]. These limitations warranted to explore a fast and efficient method for preparation of natural products from plant extract. Flash chromatography, also known as medium pressure chromatography, can efficiently overcome these limitations. It is considered as a fast, inexpensive and efficient separation technique for the separation of natural active components from extracts, and is easy to handle. This technique is an excellent alternative for slow and often inefficient gravityfed chromatography. Compared to traditional gravity-fed
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chromatography, flash chromatography gives a greatly improved reproducibility, higher resolution and recovery yield, and a reduced consumption of organic solvents. Moreover, with a UV detector and an automatic fraction collector, the sample solvent could be collected according to chromatograms profile during elution process. The aim of this work was to establish a simple and efficient protocol for preparation of biochanin A and genistein from D. odorifera leaves using macroporous resin followed by flash chromatography. Up to now, there has never been the report on separation of biochanin A and genistein by flash chromatography. The operating parameters of macroporous resin and flash chromatography were optimized. Hopefully, this work is helpful for the scale-up application for the production of biochanin A and genistein from the D. odorifera leaves and other plants.
2. Materials and methods 2.1. Materials, chemicals and adsorbents The Dalbergia odorifera T. Chen leaves were collected from Hainan Province China, and authenticated by Professor Shaoquan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, China. The leaves were dried in shade at room temperature, powdered by a disintegrator and then stored in dark. Reference compounds biochainin A (40 ,5,7-trihydroxyisoflavone-7-glucoside,P98%) and genistein (40 ,5,7-trihydroxyisoflavone,P98%) were purchased from Fluka (Buchs, Switzerland). Reagents of HPLC grade including methanol and formic acid were purchased from J&K Chemical LTD. (Beijing, China). Deionized water was produced by a Millipore Direct-Q purification system (Millipore Corp., Bedford, MA, USA). Ethanol of analytical grade was obtained from Tianjin Chemical Reagents Co. (Tianjin, China). The solvents (n-hexane, ethyl acetate, chloroform and petroleum) used for preparative flash chromatography were of analytical grade. Fourteen macroporous resins including AB-8, ADS-5, ADS-11, D101, FL-1, FL-2, FL-3, HPD500, HPD826, HPD-D, ME-2, AL-2, SA-3 and NKA-9 were purchased from Nankai Hecheng S&T (Tianjin, China) and Bonchem (Hebei, China). Their polarities ranged from non-polar to strong polar. All the resins were pretreated according to the manufacturers’ recommendation prior to use in order to remove the monomers and porogenic agents trapped inside the pores during the synthesis process [4,21]. The moisture contents of the tested resins were determined by drying the beads at 100 °C to constant weight in a drying oven for over 24 h. Toyopearl HW-40S gel resin and silica gel (300–400 mesh) were purchased from Tosoh Corporation (Tokyo, Japan) and Qingdao Meigao Chemical Co. Ltd., (Qingdao, China), respectively. TLC (Kieselgel GF254) was purchased from Taizhou Luqiao Sijia Biochemical Plastic Factory (Taizhou, China).
2.3. HPLC analysis of biochanin A and genistein Biochanin A and genistein were analyzed using an Agilent 1200 HPLC system. Chromatographic separation was carried out on a HIQ Sil C18V reversed-phase column (250 mm 4.6 mm i.d., 5 lm). All samples were filtered through 0.45 lm nylon membranes prior to HPLC analysis. The mobile phase consisted of acetonitrile (A) and water–formic acid (B). The gradient elution program was as follows: 0–5 min, 5–10% A; 5–10 min, 10–28% A; 10– 30 min, 28–60% A; 30–35 min, 60% A; 35–37 min, 60–80% A; 37– 42 min, 80–100% A; 42–55 min, 100% A. The injection volume was 5 lL. The flow rate and column temperature was 1 mL/min and 30 °C, respectively. Biochanin A and genistein were quantified at wavelength 262 nm. The chromatographic peaks of the analytes were confirmed by comparing their retention times and UV spectrum with those of the reference compounds. Eight experimental points were employed for establishing a calibration curve. The regression lines for biochanin A and genistein were Y = 84 698 x + 78.7 (R2 = 0.9949) and Y = 71 148 x + 1.37 (R2 = 0.9927), where Y is the peak area of analyte, and x is the concentration of reference compound (mg/mL). 2.4. Enrichment of biochanin A and genistein by macroporous resin 2.4.1. Screening of macroporous resins Macroporous resins can selectively adsorb and desorb constituents from sample solutions due to their specific physical and chemical properties. The adsorption and desorption capacity of different resins towards biochanin A and genistein were investigated. The adsorption tests were performed as follows: pre-weighed hydrated resins (equal to about 1.0 g dry resin) and 100 mL sample solution were added into 250 mL flasks with stopper. The flasks were shaken in an incubation shaker (120 rpm) for 4 h at 25 °C. After adsorption, the solutions were separated from the resins and analyzed by HPLC. Then, the resins were subsequently desorbed with 100 mL 80% ethanol solution. The flasks were continually shaken (120 rpm) for 6 h at 25 °C. The contents of biochanin A and genistein in desorption solutions were determined by HPLC. The adsorption properties of resins were evaluated based on the adsorption and desorption capacities and ratio of desorption. The equations were as follows: Adsorption evaluation:
Qe ¼
Pulverized D. odorifera leaves (5 kg) were extracted with 30 L of 80% ethanol at room temperature for 3 days, repeated twice. The filtered solutions were gathered and concentrated to dryness by removing the ethanol solvent using a rotary evaporator device (RE52AA, Shanghai Huxi Instrument Co., China) at 40 °C. The dried extracts were obtained, and dissolved in 30% ethanol to get sample solution (5 mg extract/mL) at the concentration of 0.2058 mg/mL for biochanin A and 0.0530 mg/mL for genistein, respectively (Table 1).
ðC 0 C e ÞV i W
where Qe is the adsorption capacity at adsorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of solute in the solutions, respectively (mg/mL); Vi is the volume of sample solution, and W is the weight of the dry resin. Desorption evaluation:
D¼
Cd V d 100% ðC 0 C e ÞV i
Qd ¼ 2.2. Preparation of D. odorifera leaves extracts
311
CdV d W
where Qd is the desorption capacity after adsorption equilibrium (mg/g resin); D is the desorption ratio (%); Cd is the concentration of solute in the desorption solution (mg/mL); Vd is the volume of the desorption solution (mL); C0, Ce, W and Vi are the same as described above. 2.4.2. Determination of macroporous resins/sample ratio A series of adsorptions was carried out with different macroporous resins/sample ratios (2:1, 3:1, 4:1, 4.5:1 and 5:1, w/w) to determine the optimal macroporous resins/sample ratio.
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Table 1 The purities and yields of the compounds in proposed separation procedure. Compound
Procedure
Purity (%)
Yield (%)
Total yield (%)
Biochanin A
Crude extract Macroporous resin enrichment Separation by silica gel flash chromatography Crude extract Macroporous resin enrichment Separation by silica gel flash chromatography
4.12 27.20 >95
— 87.13 80.13
69.82
1.06 6.79 >95
— 84.60 73.11
61.85
Genistein
Pre-weighed resins were added to the sample solution. The mixture was shaken in an incubation shaker (120 rpm) for 4 h at 25 °C. After adsorption, the solutions were separated from the resins and analyzed by HPLC. The adsorption ratios of biochanin A and genistein were calculated according to the following equation:
E¼
C0 Ce 100% C0
where E is the adsorption ratio (%), which is the percent of the adsorbed quantity to the initial quantity under equilibrium; C0 and Ce are the initial and equilibrium concentrations of solute in the solutions, respectively (mg/mL). After determining the adsorption resin and resin/sample ratio based on the above experiments, scaled-up enrichment of
Fig. 1. The actual flash chromatography system (A), schematic representation of the flash chromatography system (B), and schematic diagram of purification process for biochanin A and genistein from D. odorifera leaves (C). a – mobile phase; b – pump; c – chromatographic column; d – UV detector; e –automatic collector; f – data acquisition system.
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Fig. 2. Adsorption capacities and desorption ratios of biochanin A (A) and genistein (B) on different macroporous resins.
biochanin A and genistein was carried out to obtain enriched product for flash chromatography experiment.
TLC, the eluate was divided into three fractions: fraction A (Fr.A) containing biochanin A; fraction B (Fr.B) containing biochanin A and genistein, and fraction C (Fr.C) containing genistein.
2.5. Separation and purification of biochanin A and genistein by flash chromatography The separation and purification of biochanin A and genistein were performed on a commercial flash chromatography system (Leisure, Shanghai, China) equipped with a binary liquid pump, a UV detector and an automatic collector. The actual flash chromatography system and schematic representation of the flash chromatography system were shown in Fig. 1A and B. The enriched product of biochanin A and genistein was dissolved in methanol. The same amount of silica gel (300– 400 mesh) was added to the sample solutions, and then removed solvent by rotary evaporator device at 40 °C. The mixture of enriched product and silica gel was loaded on a column (26 mm 125 mm I.D.) which was pre-packed with silica gel (300–400 mesh). Separation of biochanin A and genistein was achieved using gradient elution system. The eluate was fractionated and collected by automatic collector according to the chromatograms at 262 nm. Based on the chromatograms profile and
Fig. 3. Effect of macroporous resins/sample ratio on the adsorption ratios of biochanin A and genistein.
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Fig. 4. HPLC chromatograms of crude extract (A), enriched product with AL-2 resin (B) and purified biochanin A (C) and genistein (D).
Then, the fraction A, B and C were evaporated by rotary evaporation, respectively. The fraction A and C were analyzed by HPLC to determine the purities and recovery yields of obtained biochanin A and genistein, respectively. In order to screen optimum elution system, three elution systems, n-hexane–ethyl acetate, n-hexane– chloroform, n-hexane–acetone, were used as gradient elution solvent. The optimum sample/silica gel ratio and flow rate were also studied systematically in this work. Another chromatogram column (26 mm 125 mm I.D.) was packed with Toyopeal HW-40S gel resin. The enriched product of biochanin A and genistein (1.0 g) was dissolved in 6 mL methanol, and then loaded on the Toyopeal HW-40 gel resin column. The biochanin A and genistein were eluted with pure methanol. The flow rate was kept at 30 mL/min. The schematic diagram of purification process for biochanin A and genistein from D. odorifera leaves was shown in Fig. 1C.
3. Results and discussion 3.1. Enrichment by macroporous resin 3.1.1. Screening of macroporous resins To enrich effectively biochanin A and genistein from D. odorifera leaves extracts by macroporous resins, the optimum type of macroporous resin was screened firstly. The properties of these resins were compared in terms of their adsorption and desorption capacities as well as desorption ratios. As can be seen from Fig. 2, the adsorption capacities of FL-1, HPD-500 and SA-3 resins towards genistein were higher, and the adsorption capacities of FL-1, ME2 and HPD-500 resins towards biochanin A were better. The adsorption capacity not only correlates with the physical and chemical properties of adsorbent, but also with the size and chemical features of the adsorbed substance. However, their lower
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desorption capacities resulted in a lower desorption ratio. Desorption ratios of AL-2 resin for genistein and biochanin A were obviously higher than those of others, and AL-2 resin possessed moderate adsorption capacity towards genistein and biochanin A. Therefore, AL-2 resin was chosen as the optimum resin for the enrichment. 3.1.2. Effect of macroporous resin /sample ratio on adsorption ratio The effect of macroporous resins/sample ratio on the adsorption ratio was investigated at macroporous resins/sample ratio of 2:1– 5:1. The results are shown in Fig. 3. It can be seen that the adsorption ratios increased with the macroporous resins/sample ratio. When the macroporous resins/sample ratio is 3:1, the adsorption ratio of bochanin A reached 90%. But in this case, the adsorption ratio of genistein was till lower. When the macroporous resins/sample ratio is 5:1, the adsorption ratios of bochanin A and genistein reached 97.45% and 90.24%. Considering simultaneous enrichment of biochanin A and genistein, a macroporous resins/sample ratio of 5:1 was chosen. In order to obtain enriched product for the following flash chromatography experiment, scaled-up enrichment of biochanin A and genistein was carried out. The chromatograms of the samples before and after treatment with AL-2 resin were shown in Fig. 4A and B. The contents of biochanin A and genistein in crude extracts were 4.12% and 1.06%. After the enrichment on AL-2 resin, the contents of biochanin A and genistein in the enriched product reached 27.20% and 6.79%, which were 6.60-fold and 6.41-fold to those in crude extract, respectively, and the recovery yields were 87.13% and 84.60%, respectively (Table 1). This process achieved easy and effective enrichment of biochanin A and genistein by using AL-2 resin.
315
resin which is based on hydroxylated methacrylic polymer [22]. Recently, due to high mechanical strength, good chemical stability and much better regeneration efficiency, it has been more and more widely applied in the separation and purification of natural products [22–25]. Especially, it exhibited potential capacity for separation and purification of flavonoids [22,23]. Fig. 5A showed the chromatogram profiles of separating biochanin A and genistein by silica gel flash chromatography. Separation of biochanin A and genistein was achieved by the following elution program: 0– 13.5 min, n-hexane–ethyl acetate (10:1, v/v); 13.5–45 min, n-hexane–ethyl acetate (6:1, v/v). The flow rate was kept at 30 mL/min. The sample/silica gel ratio was 1:40 (1.0 g enrichment product: 40 g silica gel). Based on the chromatograms profile and TLC, we collected about 21–28 min and 32.5–38 min as Fr. A and C to yield biochanin A and genistein, respectively. After HPLC analysis, the
3.2. Separation and purification of biochanin A and genistein by flash chromatography 3.2.1. Selection of stationary phase Stationary phase played a decisive role in separation of biochanin A and genistein. Two stationary phases, silica gel and Toyopeal HW-40S, were tested in this experiment. Toyopeal HW-40S gel resin, as one of the novel stationary phase, is a molecular sieve gel
Fig. 5. Chromatogram profiles of enriched product by silica gel flash chromatography at 30 mL/min (A), Toyopeal HW-40S gel resin flash chromatography at 30 mL/ min (B).
Fig. 6. Effects of elution system (A), sample/silica gel ratio (B) and flow rate (C) on the yields of biochanin A and genistein.
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were separated by using n-hexane/ethyl acetate (6:1, v/v). Hence, n-hexane–ethyl acetate was used in silica gel flash chromatography for the separation and purification of biochanin A and genistein.
Fig. 7. Chromatogram profile of enriched product by silica gel flash chromatography under optimized conditions.
purities of biochanin A and genistein were more than 95%, the recovery yields reached 76.02% and 70.52%, respectively. Biochanin A and genistein with weak-polarity was hardly eluted on Toyopeal HW-40S column by aqueous methanol. So, pure methanol was employed as mobile phase. The flow rate was kept at 30 mL/min. As shown in Fig. 5B, biochanin A and genistein were not effectively separated. Only small amounts of Fr.A and C were obtained. Although the purities of purified biochanin A and genistein were more than 95%, their recovery yields were less than 15%. Hence, the silica gel flash chromatography was suitable for preparation of biochanin A and genistein from D. odorifera T. Chen leaves. The operating parameters of silica gel flash chromatography were optimized in the following experiments. 3.2.2. Effects of elution system Biochanin A and genistein are weak-polar compounds, so low polar solvents were required to separate on normal phase silica gel column by flash chromatography. Three elution systems with weak polarity (n-hexane–ethyl acetate, n-hexane–chloroform, nhexane–acetone) were tested. The results were shown in Fig. 6A. It was found only a little Fr.A and C were obtained using n-hexane–chloroform (CHCl3) and n-hexane–acetone systems because biochanin A and genistein could not be completely separated. Although the content of biochanin A and geinstein in Fr.A and Fr.C reached more than 90%, the recovery yields of biochanin A and genistein were extremely low (less than 45%). For n-hexane– ethyl acetate system, weak-polar impurities were effectively removed when n-hexane–ethyl acetate (10:1, v/v) elution system was employed. Biochanin A and genistein with similar polarity
3.2.3. Effects of sample/silica gel ratio High sample load may distort peak shape and cause an overall decrease in separation efficiency due to column overload. So, the influence of sample load on separation efficiency was also investigated by varying the sample/silica gel ratio from 4:40 to 1:40. The elution program was the same as Section 3.2.1. The results were shown in Fig. 6B. The yields of biochanin A and genistein increased drastically with decreasing sample/silica gel ratio from 4:40 to 2:40, but did not continue to significantly increase when the sample/silica gel ratio was below 1.3:40. Although the purity of biochanin A was more than 95% at the high sample/silica gel ratio (4:40, 2:40), the recovery yield of biochanin A was remarkably low. The same trend appeared in the recovery yield of genistein. As expected, when the sample/silica gel ratio decreased, separation efficiency gradually increased due to less sample load. So, satisfactory separation of biochanin A and genistein could be achieved with a sample/silica gel ratio of 1.3:40. 3.2.4. Effects of flow rate Flow rate, as a parameter of flash chromatography, effects the retention times, system pressure and separation efficiency. In order to keep the same volume of elution solvent, elution time was adjusted with different flow rate. Fig. 6C described the effect of flow rate on separation of biochanin A and genistein by silica gel flash chromatography. As shown in Fig. 6C, the recovery yields of biochanin A and genistein gradually increased with increasing flow rate from 30 to 50 mL/min. When the flow rate is lower, the time of contact between stationary phase and target compounds is longer, leading to difficulty in elution of target compounds from the chromatography column. When flow rate was increased to 70 mL/min, the recovery yields of biochanin A and genistein decreased apparently. The separation efficiency of chromatogram column is related to its plate number. Plate number rises firstly and then declines with increasing the flow rate. In a word, excessively high flow rate has negative effect on column chromatography. Moreover, higher flow rate resulted in increase of backpressure, which might require special facilities in industrial production, leading to increase of
Fig. 8. Negative ESI–MS and MS/MS spectra of purified biochanin A (A and B) and genistein (C and D) from D. odorifera leaves, respectively.
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instrument and consumable costs [26]. These factors become a greater concern in large-scale produce. So, a flow rate of 50 mL/ min was considered as optimal one. The corresponding gradient program was: 0–8 min, n-hexane–ethyl acetate (10:1, v/v); 8– 35 min, n-hexane–ethyl acetate (6:1, v/v). 3.2.5. Validation of Fash chromatography The enriched product of biochanin A and genistein (1.3 g) was dissolved in methanol. The same amount of silica gel (300– 400 mesh) was added to the sample solutions, and then removed solvent by rotary evaporator device at 40 °C. The mixture of enriched product and silica gel was loaded on a pre-packed 40 g silica gel (300–400 mesh) column (26 mm 125 mm I.D.). Separation of biochanin A and genistein was achieved using n-hexane and ethyl acetate as gradient elution solvent. The gradient program was: 0– 8 min, n-hexane–ethyl acetate (10:1, v/v); 8–35 min, n-hexane– ethyl acetate (6:1, v/v). The flow rate was kept at 50 mL/min. The chromatogram profile of enriched product by flash chromatography was shown in Fig. 7. Under the optimized conditions, 282.98 mg biochanin A and 64.52 mg genistein were obtained by one flash chromatography run. The purities of biochanin A and genistein both reached more than 95%, and their recovery yields were 80.13% and 73.11%, respectively (Table 1). HPLC chromatograms of purified biochanin A and genistein were shown in Fig. 4C and D, respectively. The proposed silica gel flash chromatography boasts high efficiency, low running costs and process automation, and it is a promising method for efficient preparation of biochanin A and genistein from D. odorifera T. Chen leaves. 3.3. Confirmation of biochanin A and genistein The structures of purified compounds were confirmed by MS spectra. ESI–MS spectra of purified biochanin A and genistein in Fig. 8A and C showed a molecular ion [M–H] at m/z 282.9 and 269.0, respectively. The MS/MS spectra of purified biochanin A and genistein in Fig. 8B and D were also consistent with those from previous publications [27]. 4. Conclusions In this work, AL-2 was used for effective enrichment of biochanin A and genistein from extract of D. odorifera leaves. After treatment with AL-2 resin, the contents of biochanin A and genistein in the enriched product reached 27.20% and 6.79% with recovery yields of 87.13% and 84.60%, respectively. Under the optimal conditions of flash chromatography, biochanin A and genistein were successfully separated after one flash chromatography run, and their purities were over 95% with the recovery yields of 80.13% and 73.11%. The total recovery yields of biochanin A and genistein reached 69.82% and 61.85%, respectively. These results indicated that the enrichment of macroporous resin followed by the separation of flash chromatography is an effective procedure for preparing high-purity biochanin A and genistein with advantages of high recovery yield, high efficiency, low running costs and process automation. This work provides a promising alternative for large-scale preparation of flavonoids from D. odorifera leaves or other plants. Acknowledgements The authors gratefully acknowledge the financial supports by Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Importation of International Advanced Forestry Science and Technology, National
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Forestry Bureau (2012-4-06) and Heilongjiang Province Science Foundation for Excellent Youths (JC200704). References [1] R.M. Uckoo, G.K. Jayaprakasha, B.S. Patil, Rapid separation method of polymethoxyflavones from citrus using flash chromatography, Sep. Purif. Technol. 81 (2011) 151–158. [2] Y. Zhang, J.J. Jiao, C.M. Liu, X.Q. Wu, Y. Zhang, Isolation and purification of four flavone C-glycosides from antioxidant of bamboo leaves by macroporous resin column chromatography and preparative high-performance liquid chromatography, Food Chem. 107 (2008) 1326–1336. [3] T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, a specific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem. 262 (1987) 5592–5595. [4] A. Rusin, Z. Krawczyk, G. Grynkiewicz, A. Gogler, J.Z. Puchalka, W. Szeja, Synthetic derivatives of genistein, their properties and possible applications, Acta Biochim. P. 57 (2010) 23–34. [5] C.E. Park, H. Yun, E.B. Lee, B.I. Min, H. Bae, W. Choe, I. Kang, S.S. Kim, J. Ha, The antioxidant effects of genistein are associated with AMP-activated protein kinase activation and PTEN induction in prostate cancer cells, J. Med. Food 13 (2010) 815–820. [6] Y. Zhang, W.F. Chen, W.P. Lai, M.S. Wong, Soy isoflavones and their bone protective effects, Inflammopharmacol 16 (2008) 213–215. [7] V. Sehdev, J.C.K. Lai, A. Bhushan, Biochanin a modulates cell viability invasion and growth promoting signaling pathways in HER-2-positive breast cancer cells, J. Oncol. (2009) 121458. [8] P. Mishra, A. Kar, R.K. Kale, Modulatory influence of prepubertal biochanin a exposure on mammary gland differentiation and expression of estrogen, receptor-a and apoptotic proteins, Phytother. Res. 23 (2009) 972–979. [9] D.Y. Zhang, Y.G. Zu, Y.J. Fu, M. Luo, C.B. Gu, W. Wang, X.H. Yao, Negative pressure cavitation extraction and antioxidant activity of biochanin A and genistein from the leaves of Dalbergia odorifera T Chen, Sep. Purif. Technol. 83 (2011) 91–99. [10] J.R. Kumar, H.I. Lee, J.Y. Lee, J.S. Kim, J.S. Sohn, Comparison of liquid–liquid extraction studies on platinum(IV) from acidic solutions using bis(2,4,4trimethylpentyl) monothiophosphinic acid, Sep. Purif. Technol. 63 (2008) 184– 190. [11] L.H. Wang, Y.H. Mei, F. Wang, X.S. Liu, Y. Chen, A novel and efficient method combining SFE and liquid–liquid extraction for separation of coumarins from Angelica dahurica, Sep. Purif. Technol. 77 (2011) 397–401. [12] P. Priyananda, V. Chen, Separation of residual fatty acids from aqueous solutions using an agitated solution of protein and membrane filtration, Sep. Purif. Technol. 48 (2006) 113–120. [13] R.M.F. Espinosa, H.B.O. Oliveros, M.T. Olguín, M.R.P. Cueto, R.G.S. Vicente, Separation and treatment of ion-exchange resins used in cleaning systems of a research nuclear reactor, Chem. Eng. J. 188 (2012) 71–76. [14] G. Favaro, F. Magno, A. Boaretto, L. Bailoni, R. Mantovani, Traceability of asiago mountain cheese: a rapid, low-cost analytical procedure for its identification based on solid-phase microextraction, J. Dairy Sci. 88 (2005) 3426–3434. [15] Y.F. Liu, J.X. Liu, X.F. Chen, Y.W. Liu, D.L. Di, Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins, Food Chem. 123 (2010) 1027–1034. [16] M.R. Kim, W.C. Kim, D.Y. Lee, Recovery of narirutin by adsorption on a nonionic polar resin from a water-extract of Citrus unshiu peels, J. Food Eng. 78 (2007) 27–32. [17] A. Schieber, P. Hilt, H.U. Endress, A new process for the combined recovery of pectin and phenolic compounds from apple pomace, Innovative Food Sci. Emerg. Technol. 4 (2003) 99–107. [18] S. Liu, L.Z. Yi, Y.Z. Liang, Traditional Chinese medicine and separation science, J. Sep. Sci. 31 (2008) 2113–2137. [19] X.J. Li, Y.J. Fu, M. Luo, W. Wang, L. Zhang, C.J. Zhao, Y.G. Zu, Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography, J. Pharm. B. 61 (2012) 199–206. [20] Z.F. Wei, Y.G. Zu, Y.J. Fu, W. Wang, C.J. Zhao, M. Luo, T. Efferth, Resin adsorption as a means to enrich rare stilbenes and coumarin from pigeon pea leaves extracts, Chem. Eng. J. 172 (2011) 864–871. [21] B.Q. Fu, J. Liu, H. Li, L. Li, F.S.C. Lee, X.R. Wang, The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid, J. Chromatogr. A 1089 (2005) 18–24. [22] L.U. Hansen, M. Yanagishitag, Differential effect of brefeldin a on the biosynthesis of heparan sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture, J. Biol. Chem. 268 (1993) 17370–17376. [23] L. Liu, B.J. Xie, S.Q. Cao, E.N. Yang, X.Y. Xu, S.S. Guo, A-type procyanidins from Litchi chinensis pericarp with antioxidant activity, Food Chem. 105 (2007) 1446–1451. [24] S.Q. Cao, S.Y. Pan, X.L. Yao, H.F. Fu, Isolation and purification of anthocyanins from blood oranges by column chromatography, Agric. Sci. China 9 (2010) 207–215. [25] B. Hu, L. Wang, B. Zhou, X. Zhang, Y. Sun, H. Ye, L.Y. Hao, Q.H. Hu, G.X. Wang, X.X. Zeng, Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine
318
F.-Y. Ma et al. / Separation and Purification Technology 120 (2013) 310–318
alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection, J. Chromatogr. A 1216 (2009) 3223–3231. [26] S.H. Pyo, H.J. Choi, An improved high-performance liquid chromatography process for the large-scale production of paclitaxel, Sep. Purif. Technol. 763 (2011) 78–384.
[27] R. Vessecchi, G.J. Zocolo, D.R. Gouvea, F. Hübner, B. Cramer, M.R.R. de Marchi, H.U. Humpf, N.P. Lopes, Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies, Rapid Commun. Mass Spectrom. 25 (2011) 2020–2026.