314 Jianyong Feng1∗ Xihui He2∗ Sheng Zhou1 Fang Peng1,2 Jiangyun Liu1 Lili Hao1 Heran Li1 Guizhen Ao1 Shilin Yang1 1 College
of Pharmaceutical Sciences, Soochow University, Suzhou, China 2 School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China
Received June 6, 2013 Revised October 2, 2013 Accepted November 12, 2013
J. Sep. Sci. 2014, 37, 314–322
Research Article
Preparative separation of crocins and geniposide simultaneously from gardenia fruits using macroporous resin and reversed-phase chromatography Gardenia fruits contain valuable natural food colorants including crocins (gardenia yellow) and geniposide. In this study, a process for the enrichment of crocins and geniposide simultaneously from gardenia fruits was developed using macroporous resin and RP chromatography. The performance of eight different types of macroporous resins was evaluated. Static absorption/desorption experiments revealed that LX60 possessed optimal separating capacity. Further dynamic absorption/desorption experiments on LX60 columns were conducted to obtain the optimal parameters. After one run treatment with LX60, the content of crocin-1 in gardenia yellow reached 29.6%, while geniposide in another fraction reached 83.4%. An extract of crocins was obtained from gardenia yellow in a second-stage separation using RP medium-pressure LC, with its color value to be 756 and the content of crocin-1 reaching 60.8%. The separation process was highly efficient, low cost, and compact, which may be informative for purifications of other natural products from complex plant extracts. Keywords: Crocin / Gardenia jasminoides / Geniposide / Macroporous resin / RP chromatography DOI 10.1002/jssc.201300601
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Crocins are secondary metabolites abundant both in saffron (the dried stigmas of Crocus sativus L.) and in gardenia (Gardenia jasminoides Ellis) fruits [1–3]. They are oligosaccharide esters of crocetin (8,8 -diapocarotene-8,8 -dioic acid), a special carotenoid derivate (Fig. 1). Due to their unique water-soluble and antioxidant properties, crocins are applied worldwide as a natural food colorant, mainly in colored juice, jelly, candy, and noodles [4–7]. Numerous studies have also demonstrated a variety of pharmacological actions for crocetin and crocins, including protection against cardiovascular diseases [8] and cerebral ischemia atherosclerosis [9], prevention of retinal degeneration [10], and inhibition of tumor cell proliferation [11]. As saffron is highly valued as the world’s most expensive spice [5], it is of great interest to separate crocins alternatively from gardenia fruit (gardenia yellow) to meet the increasing demand from international markets. Correspondence: Dr. Jiangyun Liu, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China E-mail: [email protected] Fax: +86-512-65882089
Abbreviations: BV, bed volume; MAR, macroporous adsorption resin; MPLC, medium-pressure LC; PDA, photodiode array C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Gardenia jasminoides Ellis is widely cultivated in Southeast Asia, especially in the south of China. Gardinia fruit has been used as a traditional Chinese medicine for its homeostatic, antiphlogistic, choleretic, diuretic, hemostatic, and anticancer properties [12]. Besides crocins, geniposide and other iridoid glycosides, and quinic acid derivatives (such as chlorogenic acid) (Fig. 1) are also present and well characterized in gardenia fruit [13–15]. Geniposide (the major one) and other iridoid glycosides contribute to the many functions of gardenia fruits, such as anti-inflammatory [16], immunosuppressive [17] and other bioactivities. Quinic acid derivatives are also well-known antioxidants beneficial for human health [14]. On the other hand, gardenia yellow is liable to fade and turns green in food application because of the coexistence of geniposide and quinic acid derivatives [18]. Therefore, it is critical to establish an efficient extracting process in industrial practice to separate both gardenia yellow (with high color value) and geniposide simultaneously for integrated utilization of this valuable herb. Extraction methods have been developed extensively for the separation of gardenia yellow, including multiple solvent extraction, membrane filtration, adsorption chromatography ∗ These authors contributed equally to this work. Additional corresponding author Professor Lili Hao, E-mail: [email protected]. Colour Online: See the article online to view the Fig. 3 in colour.
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
315
Figure 1. Chemical structures of crocins, geniposide, and chlorogenic acid.
methods, and imprinted polymer SPE at different process stages [19–21]. The conventional methods for the separation of crocins were performed by means of solid–liquid extraction, liquid–liquid extraction, followed by multistep silica gel column chromatography and semi-preparative HPLC [1,2,4]. However, these methods are not suitable for large-scale production due to the need of bulk amounts of organic solvents and laborious work. In recent years, macroporous adsorption resins (MARs) have been successfully applied for the enrichment of target compounds from crude extracts in industrial practices due to their high efficiency, easy regeneration, and environmentally friendly features [22–29]. Enrichments of gardenia yellow using macroporous resins have been reported, with its color value to be about 300 [23]. However, it remains difficult to separate crocins from gardenia fruits with high purity, high recovery rate, and low cost. Multiplestage separation methods, such as RP silica [24,25], polyamide [26,27], high-speed counter-current [28] chromatography, and crystallization techniques [29] are necessary to be applied in combination with macroporous resin to obtain pure ingredients that are frequently demanded in pharmaceutical applications. These methods could be applied in the further separation of crosins, which remain little reported. It is always important to analyze both target ingredients and impurities at first before a suitable preparation process can be rationally developed. An on-line HPLC with photodiode array (PDA) system has been applied as a powerful method to monitor complex ingredients at multiple wavelengths, as evidenced for HPLC analysis of gardenia fruits [13–15] as well as other traditional Chinese medicines in recent publications [30, 31]. In the present study, a simple and efficient process was developed to enrich both crocins (with color value >700) and geniposide fractions simultaneously from gardenia fruits using macroporous resins and RP medium-pressure LC (MPLC). To fully evaluate the properties of the gardenia extracts at different separation stages, an HPLC–PDA method was applied for the qualitative and quantitative analysis of crocin-1, geniposide, and quinic acid derivates at the same time. Moreover, the color value and optical density ratio value of the crocin extracts were also C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
evaluated. The separation procedure developed here could be informative for purifications of other natural products from complex plant extracts.
2 Materials and methods 2.1 Chemicals, reagents, and samples A crocin-1 standard was purified and identified by ESIMS, NMR spectroscopy, and UV adsorption spectroscopy. Geniposide was purchased from the National Institutes for Food and Drug Control. The purities of crocin-1 and geniposide were 98.7 and 99.2%, respectively, as determined by HPLC. Appropriate amounts of standards were dissolved in methanol to yield the stock solutions at the concentrations of 0.8204 mg/mL for crocin-1 and 1.033 mg/mL for geniposide. Ethanol (analytical grade), methanol (HPLC grade) and other reagents (analytical grade) were all purchased from Shanghai Chemical Reagents (Shanghai, China). Distilled water was purified by a DW100 purification system from Shanghai HiTech Instruments (Shanghai, China).
2.2 Adsorbents MARs including D101, X-5, LX60, AB-8, LX38, LX28, LX8, and LX17 were purchased from Xi’an Sunresin Technology (Shaanxi province, China). Before the adsorption experiments, the resins were weighed and pretreated by soaking in ethanol overnight, subsequently washing once by 4% HCl, distilled water, and 4% NaOH solution, respectively, and finally washing by distilled water thoroughly to remove the monomers and porogenic agents trapped inside the pores during the synthesis process. The moisture contents of the tested resins were determined by drying the beads (1.0 g in wet weight) at 80⬚C to constant weight for over 24 h. The chemical and physical properties of these MARs are summarized in Table 1. www.jss-journal.com
316
J. Sep. Sci. 2014, 37, 314–322
J. Feng et al.
Table 1. Chemical and physical properties of eight macroporous resins in test
Resin
Structure
Polarity
Specific surface area (m2 /g)
Pore size (nm)
Particle size (mm)
Moisture content (%)
D101 X-5 LX60 AB-8 LX38 LX28 LX8 LX17
Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Acrylate
Non Non Non Weak Weak Middle Polar Middle
650–700 500–600 850 480–520 600 500 – ≥460
8.5–9 7.0 7.0 13–14 5.0 7.1 – 13–14
0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25
64.8 65.1 72.6 44.5 68.9 59.4 80.2 59.7
2.3 Preparation of the crude extracts of gardenia fruits The dried fruits of Gardenia jasminoides Ellis were collected in Luzhai county, Guangxi province of China in October 2011, and were authenticated by Professor Lili Hao of Soochow University, and a voucher specimen (No. GJ20111001) was deposited there. Gardenia fruits (1.0 kg) were ground to powder, extracted with 40% aqueous ethanol (8 L) under reflux for 1.5 h, repeated twice. The extracting liquors were combined and concentrated to 2.0 L to get the crude extract solution (GJE, with the ratio of raw material to extract volume to be of 0.5 g/mL), which contained 4.120 mg/mL of crocin-1 and 18.28 mg/mL of geniposide. The sample solutions were stored at 4⬚C until use. Deionized water was added to get solutions of different concentrations.
2.4 HPLC analysis of crocin-1 and geniposide Quantitative analysis was carried out by HPLC on a Shimadzu Prominence LC-20A liquid chromatographic system (Shimadzu instruments company, Japan) composed of binary pumps, a PDA detector, and LC solution software. A Cosmosil C18 column (250 mm × 4.6 mm i.d., 5 m) was used at a column temperature of 25⬚C. The mobile phase was consisted of methanol (A) and water (B, with 0.1% acetic acid) with the following gradient program: 0–4 min, 20% A; 4–40 min, 20–90% A. The flow rate was set at 1.00 mL/min, and PDA on-line monitor wavelengths were set at 238 and 440 nm. The retention times of geniposide and crocin-1 were determined to be 15.63 (monitored at 238 nm) and 27.58 min (monitored at 440 nm), respectively. The chromatographic peaks were identified by comparing their respective retention times and UV spectra with those of reference standards, which were eluted in parallel under the same conditions. All samples were filtered through a 0.45 m membrane filter before use. The working calibration curves showed good linearity over the range of 0.8204–11.48 g for crocin-1 and 2.066– 20.66 g for geniposide. The regression curves for crocin-1 and geniposide were Y = (5.5303 × 106 ) X – 1.0221 × 106 (R2 = 0.9993, n = 6), and Y = (1.0976 × 106 ) X – 0.0461× 106 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(R2 = 0.9997, n = 6), respectively, where Y is the peak area of the analyte and X is the injected quantity of crocin-1 and geniposide (g).
2.5 UV/Vis measurement of the color value of gardenia yellow The color value of gardenia yellow was measured as reported [18]. Briefly, dried powder sample (0.15 g) was dissolved in a 100 mL volumetric flask using water as the solvent. Ten milliliters of this sample solution was then transferred to another 100 mL volumetric flask and diluted. Finally, the absorbance of the diluted solution was measured in a standard 1.0 cm pathlength cuvette by a Shimadzu UV-2600 UV/Vis spectrophotometer (Shimadzu, Japan) at 238 (max of geniposide), 328 (max of quinic acid derivates), and 440 nm (max of crocins) (absorbance should be controlled between 0.3 and 0.7). The color value (E) and optical density ratio values (OD) of gardenia yellow were calculated as follows: E = A440 /C
(1)
OD1 = A238 /A440
(2)
OD2 = A328 /A440
(3)
where E stands for the colority of gardenia yellow (A440 with sample amount of 1.0% g/mL using a standard 1.0 cm pathlength cuvette), C is the concentration of the diluted sample solution (g/100 mL), A440 , A238 , and A328 is the absorbance at 440, 238, and 328 nm, respectively.
2.6 Procedures for the batch static absorption and desorption tests 2.6.1 Static adsorption resin screening All macroporous resins were screened through static adsorption tests, which were performed as follows: The hydrated www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
resins (1.0 g dry mass) were put into an Erlenmeyer flask and 50 mL of sample solutions (0.6-fold dilution of GJE, with concentrations of 2.472 mg/mL crocin-1 and 10.97 mg/mL geniposide) were added. The flasks were then shaken (120 rpm) for 5 h at 25⬚C to reach adsorption equilibrium. The solutions after absorption were analyzed by HPLC. After reaching absorption equilibrium, the resins were first washed by deionized water and then desorbed with 50 mL of 95% ethanol. The flasks were shaken (120 rpm) for 5 h at 25⬚C. The desorbed solutions were then analyzed by HPLC. Each experiment was done in triplicate. The preliminary choices of the resins were evaluated by their capacities of static absorption/desorption and desorption ratios. The following equations were used to quantify the absorption and desorption capacities [24]. Absorption capacity: Qe = (C0 − Ce )Vi /W
(4)
where Qe represents the absorption capacity at absorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of solutes in the solutions, respectively (mg/mL); Vi is the volume of the initial feed solution (mL); and W is the weight of the dried adsorbent (g). Desorption ratio: D = Cd Vd /(C0 − Ce )Vi × 100%
(5)
where D is the desorption ratio (%); Cd is the concentration of the solutes in the effluent (mg/mL); Vd is the volume of the effluent.
2.6.2 Adsorption kinetics on LX60 resin The adsorption kinetics curves of crocin-1 and geniposide on the initially selected LX60 resins were studied according to the following process: adding appropriate pretreated resins (equal to 1.0 g dry mass) and 50 mL of sample solutions (0.6fold dilution of GJE) to each flask with a lid, and then shaking for 5 h at 25⬚C (120 rpm). The concentrations of crocin-1 and geniposide in the adsorption process were determined by HPLC at certain time intervals after pretest (5, 10, 20, 30, 45, 60, 90, 120, 180, 240, and 300 min).
2.6.3 Adsorption isotherms on LX60 resin Stock solutions were diluted to get nine different sample solutions in sequence, with concentrations between 0.2472– 4.120 mg/mL for crocin-1 and 1.097–18.28 mg/mL for geniposide. Each sample solution (50 mL) was combined with pretreated resins (equal to 1.0 g dry mass) in shakers and shaken (120 rpm) for 5 h at 25⬚C. The initial and equilibrium concentrations were determined by HPLC. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
317
2.7 Dynamic adsorption/desorption experiments 2.7.1 Dynamic adsorption/desorption on selected resins The separation properties of three selected resins (D101, LX60, and LX17) were further evaluated by the dynamic adsorption and desorption tests. The experiments were conducted in a glass column (30 × 1.5 cm id) packed with 5.5 g (dry mass) selected resins. The bed volume (BV) of the wet-packed resin was 8 mL. Sample solutions (40 mL) flowed through the column at 0.5 mL/min, and the concentration of crocin-1 was 3.296 mg/mL and geniposide was 14.62 mg/mL (0.8-fold dilution of GJE). For the separation process of the three selected resins, the adsorbents were conditioned by gradient elution with water, 20%, and 70% ethanol (each for 48 mL), respectively after adsorption equilibrium of sample solutions at the flow rate of 0.5 mL/min. Each part of the desorption solution was analyzed by HPLC and UV/Vis spectroscopy methods. Each experiment was conducted in triplicate.
2.7.2 Dynamic adsorption/desorption on LX60 resin Several critical parameters, including the concentration and volume of feed solution, the percentage of ethanol in solutions and the volume of the eluents for desorption process were systematically investigated at room temperature. The tests were conducted on a glass column (30 × 1.0 cm id) wetpacked with LX60 (equal to 2.75 g dry mass, 4 mL/BV). The adsorption process was performed by loading feed solution into the pretreated glass column. After adsorption equilibrium for more than 180 min, desorption was completed by gradient elution with solutions of water and ethanol at different ratios. To chose appreciate feed concentration and volume, four different feeding concentrations (0.4-, 0.6-, 0.8-, and 1.0-fold dilution of GJE) of sample solutions were tested. Sample solutions were fed to overload (180, 120, 100, 60 mL, respectively). The eluent fractions were collected every 4 mL and then measured by HPLC. To test the percentage of ethanol in solutions, sample solutions (0.8-fold dilution of GJE, 40 mL) were applied. After the adsorption equilibrium, the adsorbate-laden columns were washed with deionized water, 5, 10, 20, 30, 40, 50, 70, and 95% ethanol (each 24 mL) successively. Each part of eluents was combined and then analyzed by HPLC. Sample solutions (0.8-fold dilution of GJE, 40 mL) were also applied in another test to select the eluent volume needed. After the adsorption equilibrium, the adsorbateladen columns were washed with deionized water (24 mL), 20% ethanol (32 mL), 70% ethanol (36 mL), and 100% ethanol (20 mL) successively. The eluent fractions of 20 and 70% ethanol parts were collected every 4 mL and then measured by HPLC. www.jss-journal.com
318
J. Sep. Sci. 2014, 37, 314–322
J. Feng et al.
2.7.3 Scale-up enrichment of crocins and geniposide from gardenia fruits Scale-up separation was conducted by about 36-fold as that of lab levels. A glass column (45 × 3.5 cm id) was slurry packed with LX60 resin (100 g, dry weight, 150 mL/BV). Sample solution (1.5 L, corresponding to extract from 600 g gardenia fruits) of crude extract (with concentrations of 3.255 mg/mL crocin-1 and 13.83 mg/mL of geniposide) was applied to the column. After sample loading and adsorption equilibrium for more than 180 min, desorption was performed successively with water, 20% ethanol, 70% ethanol (each 900 mL) at a flow rate of 450 mL/h. The effluents of 20 and 70% ethanol parts were collected and dried to yield GJ-1 (geniposide fraction, 20.7 g) and GJ-2 (gardenia yellow fraction, 14.5 g), respectively. Sample fractions were also subjected to analytical HPLC and UV/Vis spectroscopy to determine the contents of geniposide and crocin-1 and the color values. 2.8 MPLC separation of crocins 2.8.1 Purification of crocin and geniposide compounds Separation chromatography was carried out according to Ref. [18], using a MPLC instrument with a PDA detector (BP-Purifier-100, Lisure science corporation, Suzhou, China). The column (15 × 2.5 cm id) was packed with silica gel (300– 400 mesh, Qingdao Haiyang chemical corporation, Qingdao, China) or Cosmosil C18 silica gel (75 m, Nacalai tesque corporation, Tokyo, Japan) by a simple dry-pack method. The BV was 70 mL. As for silica gel column purification, GJ-2 (1.0 g) was dissolved in methanol (10 mL), mixed with 2 g of silica gel and condensed. The dry sample was loaded on the top of the column and eluted with EtOAc/MeOH/H2 O (10:2:1, 900 mL, fraction 1–18) followed by EtOAc/MeOH/H2 O (10:3:1, 800 mL, fraction 19–32). The flow rate was set at 50 mL/min, UV/Vis detection was set at 328 and 440 nm, each fraction was collected at 50 mL, and all the chromatographic experiments were conducted at ambient temperature. The fractions corresponding to crocin-1 (78 mg, fraction 26– 29) were collected and subjected to HPLC to determine the content and the product recovery rate. As for RP separation, C18 column was washed with 100% methanol extensively before use, and then pre-equilibrated with 43% methanol. GJ-2 (1.0 g) was dissolved in 43% methanol (10 mL) and loaded into the column by an injection syringe, eluted with 43% methanol (1.5 L, fraction 1–30), followed by 60% (150 mL, fraction 31–33), 70% (100 mL, fraction 34–35), and 100% (150 mL, fraction 36–38) methanol. The flow rate was set at 50 mL/min, UV detection was set at 328 and 440 nm, and all the chromatographic experiments were conducted at ambient temperature. The peaks corresponding to quinic acid derivatives or crocins were collected and subjected to analytical HPLC to determine the content and the C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
product recovery rate, respectively. Trace amount of geniposide was detected in fractions 3 and 4. Quinic acid derivatives were detected in fractions 5–22. Crocin-1 (164 mg), crocin-2 (19 mg), and crocin-3 (51 mg) were obtained from fractions 24–28, 32–33, and 35, respectively, and identified by comparison of their NMR and UV spectroscopic data with published data [1, 2, 32]. GJ-1 (1.0 g) was dissolved in hot EtOAc (20 mL) and filtered, cooled to room temperature and then filtered again to give geniposide (460 mg). Geniposide was identified by comparisons of its retention time in HPLC and its UV spectrum with a reference standard. 2.8.2 Preparative separation of crocins by RP MPLC For the preparative separation of crocins, an RP column (25 × 3.5 cm id) packed with Cosmosil C18 silica gel (75 m) was applied. The BV was 240 mL. The column was pre-equilibrated with 40% methanol. GJ-2 (10.0 g) was dissolved in 40% methanol (100 mL) and loaded into the column by an injection syringe, eluted with 40% methanol (3.0 L) to remove impurities, followed by methanol (1.0 L). The flow rate was set at 100 mL/min, UV detection was set at 328 and 440 nm, and all the chromatographic experiments were conducted at ambient temperature. The peaks corresponding to crocins (GJ-3, 4.76 g) or quinic acid derivatives (GJ-4, 3.41 g) were collected and subjected to analytical HPLC to determine the content and the product recovery rate, respectively.
2.9 Statistical analysis Data were expressed as means ± standard error of three separate experiments, and a t-test was used for determining the statistically significant differences (P < 0.05).
3 Results and discussion 3.1 The preliminary choice of the resins 3.1.1 Batch static adsorption/desorption experiments Adsorption characteristics of macroporous resins are not only closely related to polarity and size of adsorbates, but also to dimensional structure (specific surface area, pore diameter, and pore volume) of adsorbents, and to the adsorption medium. Eight typical macroporous resins (Table 1) were therefore selected and tested for their adsorbing and separation performance. The results of static adsorption tests and desorption are shown in Table 2. MARs in this survey demonstrated different adsorption capacities, ranging from 34.3–95.4 mg/g for crocin-1 and 75.9–186.8 mg/g for geniposide. By comparison of different polystyrene resins, three nonpolar resins www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
319
Table 2. Adsorption capacities, desorption capacities, and desorption ratios of eight resins for crocin-1 and geniposide
Resin
D101 X-5 LX60 AB-8 LX38 LX28 LX8 LX17
Adsorption (mg/g)
Desorption (mg/g)
Crocin-1
Geniposide
92.1 ± 90.2 ± 95.4 ± 56.1 ± 58.9 ± 58.2 ± 34.3 ± 89.4 ±
134.2 152.5 186.8 120.7 99.7 75.9 89.8 161.6
1.9 4.1 3.6 1.2 3.5 2.9 1.7 3.8
± ± ± ± ± ± ± ±
2.7 3.2 3.1 1.3 1.8 0.9 2.1 3.6
Desorption ratio (%)
Crocin-1
Geniposide
78.4 ± 75.3 ± 88.7 ± 48.2 ± 53.8 ± 46.8 ± 25.5 ± 75.8 ±
106.2 133.0 168.1 111.3 84.8 56.9 80.5 138.6
2.5 3.2 3.1 3.9 4.1 1.9 0.8 2.7
(D101, X5, and LX60) showed apparent higher adsorption capacities than other weak-polar/polar resins, indicating a preferred hydrophobic interaction with the adsorbates (aglycone groups). LX60 achieved a better adsorption/desorption capacity than D101 and X5, partly because of its higher specific surface areas than that reported by Yang et al. [22]. LX17 belonging to the polyacrylate class also exhibited excellent adsorption/desorption efficiency similar to that of D101 or X5. This may be attributed to the main electrostatic attraction and hydrogen-bonding interactions between the polyacrylate resins and adsorbates (glucosyl group) [25]. Accordingly, three resins, LX60, D101, and LX17 were selected for further dynamic separation investigations.
3.1.2 Dynamic separation performance of selected resins Dynamic separation properties of three selected resins toward crocin-1 and geniposide were tested as shown in Table 3. Geniposide could be efficiently desorbed by 20% ethanol for three resins, while crocin-1 was not desorbed until 70% ethanol. Two products, geniposide (GJ-1) and gardenia yellow (GJ-2) extracts could thus be obtained in this stage. Generally, the colority of the gardenia yellow product reached 300 after purification by macroporous resin such as HPD722 [23]. The color value of two samples performed better to be 392 (for D101) and 423 (for LX60). This was in agreement with higher adsorption capacities of the resins applied as evidenced in static tests (Table 2). The efficient removal of geniposide using 20% ethanol could also contribute to higher purity of
± ± ± ± ± ± ± ±
1.4 1.6 3.3 2.5 1.1 1.2 2.9 2.1
Crocin-1
Geniposide
85.1 83.6 92.9 85.9 91.2 80.4 74.5 84.7
79.1 87.2 89.9 92.2 85.1 74.8 89.7 85.8
GJ-2. Considering of its higher adsorption capacity and purity of products, LX60 was chosen as optimal resin for the enrichment of crocin-1 and geniposide.
3.2 Static/Dynamic experiments on LX60 resin 3.2.1 Adsorption kinetics on LX60 resin The adsorption kinetics curves of crocin-1 and geniposide on LX60 are shown in Supporting Information Fig. S1A. The adsorption of crocin-1 and geniposide on LX60 resin increased with increasing adsorption time. The adsorption increased rapidly in the first 60 min, and then it increased slowly, and finally reached equilibrium after 180 min for crocin-1 and 120 min for geniposide, indicating that LX60 belongs to the slow adsorption resin type. Therefore, the batch adsorption equilibrium tests were run for over 180 min. Adsorption kinetics parameters for both crocin-1 and geniposide on LX60 were calculated (Supporting Information Table S1). A pseudo-second-order kinetics model was suitable to describe the entire adsorption process on the resin, with calculated correlation coefficient (R2 ) to be 0.9923/0.9986 for both adsorbates. The pseudo-second-order kinetics implied that concentrations of both adsorbates and adsorbents were involved in rate-determining steps during the adsorption process. These results indicated that the adsorption process was controlled by two or more rate-limiting steps such as external diffusion, boundary layer diffusion, and intra-particle diffusion [24].
Table 3. Dynamic adsorption capacities and properties of crocin-1 and geniposide on three selected resins
Resin
D101 LX60 LX17
Adsorption (mg/g)
Content (%)
Crocin-1
Geniposide
Crocin-1
Geniposide
Crocin-1
Geniposide
E
OD1
OD2
49.7 ± 2.3 54.5 ± 1.9 48.4 ± 2.1
121.5 ± 3.1 156.3 ± 3.5 113.5 ± 3.9
23.0 ± 0.7 25.6 ± 0.9 24.5 ± 1.1
79.7 ± 1.6 83.4 ± 1.3 80.8 ± 0.5
80.6 82.4 79.7
73.6 75.7 68.2
392 423 347
0.420 0.407 0.512
0.409 0.385 0.601
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Recovery (%)
Color value
www.jss-journal.com
320
J. Feng et al.
3.2.2 Adsorption isotherms on LX60 resin Equilibrium adsorption isotherms of LX60 resin were measured at 25⬚C as shown in Supporting Information Fig. S1B. The adsorption capacity of LX60 resin toward both crocin-1 and geniposide increased as the adsorbate concentrations increased, and reached saturation at 3.296 mg/mL for crocin-1 and 14.62 mg/mL for geniposide, respectively. The results provided reference values for scaling up the process with regard to crude extract concentrations prior to the chromatography. To simulate the equilibrium adsorption behaviors, Langmuir and Freundlich parameters were calculated and summarized in Supporting Information Table S2. The Langmuir equation described the adsorption behavior of crocin-1 and geniposide on LX60 resin better because the calculated correlation coefficients (0.9924 and 0.9946, respectively) of the Langmuir equations were higher than that of the Freundlich equations. These results suggested the monolayer coverage of crocin-1 and geniposide onto the resin. The theoretical maximum adsorption capacity (Qmax ) of crocin-1 (geniposide) determined from the Langmuir equation was 91.74 mg/g (188.7 mg/g). In the Freundlich equation, the adsorption is very difficult to achieve if 1/n value is above 2 [24]. In Supporting Information Table S2, the 1/n values are 0.2366 and
J. Sep. Sci. 2014, 37, 314–322
0.3215, respectively, which indicates that the LX60 resin is suitable for separating crocin-1 and geniposide.
3.2.3 Dynamic adsorption and desorption tests on LX60 resin 3.2.3.1 Effect of feeding concentration and volume In the process of dynamic adsorption, the effects of feeding concentration and feeding volume on adsorption capacity were investigated, and the results of leakage curve at different concentrations were obtained (Fig. 2A and B). The adsorption capacities slightly increased but fell back with the increment of initial feed concentrations due to competition of active sites of LX60 resins by impurities in the crude extracts and therefore limiting the diffusion of crocin-1 into the micropores of LX60. The breakthrough point generally refers to the circumstance under which the adsorbate concentration in the eluent reaches 5% of that in the sample solution applied. In the present study, the adsorption capacity did not increase apparently when the initial concentration of crocin-1 (geniposide) was >3.296 mg/mL (14.62 mg/mL), and the corresponding feed volume was 40 mL (with ratio of corresponding raw material and resin dry mass to be 5.8 g/g).
Figure 2. Effects of feed concentration (A) and volume (B), and elution solvent effects of ethanol concentration (C) and volume (D) on content of crocin-1 and geniposide.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
321
Table 4. Contents and color values of crocin-1 and geniposide in different extracts
Extract
Content (%)
GJE
0.8601a) 3.806b) 83.38b) 29.65a) 60.78a)
GJ-1 GJ-2 GJ-3
Yield (%)
3.45 2.41 1.13
Recovery (%)
75.7 83.1 81.2
E
OD1
OD2
67
1.831
0.517
390 756
0.421 0.148
0.375 0.142
Note: a)Crocin-1; b)Geniposide.
3.3 Scale-up preparation of crocins and geniposide on LX60 resin
Figure 3. HPLC–PDA chromatograms of different fractions of gardenia fruits monitored at 440 (I), 238 (II), and 328 (III) nm simultaneously. (A) Mixture solution of crocin-1 and geniposide reference substances; (B) GJE (crude extract); (C) GJ-1 (geniposide) fraction; (D) GJ-2 (gardinia yellow) fraction; (E) GJ-3 (crocin) fraction; (F) GJ-4 (quinic acid derivates) fraction. 1 geniposide; 2 crocin-1.
3.2.3.2 Effect of the eluent concentration and volume on desorption In the process of dynamic adsorption, the effect of ethanol concentration for the desorption process was investigated. After adsorption equilibrium, the adsorbate-laden column was first washed with deionized water, and then desorbed by gradient elutions with 5–95% ethanol, respectively. Figure 2C showed that geniposide could be desorbed mainly with 5–30% ethanol, and crosin-1 could be desorbed after 20% ethanol. The procedure was thus set by elution with water at first to remove impurities, followed by 20% (to afford geniposide fraction) and 70% (to desorb crocins efficiently) ethanol. Regarding the eluent consumption of the separation process as an important factor, the volume of eluent was investigated under the optimal separation conditions. The concentrations of crocin-1 and geniposide in the eluent were plotted against eluent volumes, and the elution curves were obtained as shown in Fig. 2D. The concentrations of both analytes in the eluent reached maximum at the third fraction, then decreased rapidly, and fell to
of Pharmaceutical Sciences, Soochow University, Suzhou, China 2 School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China
Received June 6, 2013 Revised October 2, 2013 Accepted November 12, 2013
J. Sep. Sci. 2014, 37, 314–322
Research Article
Preparative separation of crocins and geniposide simultaneously from gardenia fruits using macroporous resin and reversed-phase chromatography Gardenia fruits contain valuable natural food colorants including crocins (gardenia yellow) and geniposide. In this study, a process for the enrichment of crocins and geniposide simultaneously from gardenia fruits was developed using macroporous resin and RP chromatography. The performance of eight different types of macroporous resins was evaluated. Static absorption/desorption experiments revealed that LX60 possessed optimal separating capacity. Further dynamic absorption/desorption experiments on LX60 columns were conducted to obtain the optimal parameters. After one run treatment with LX60, the content of crocin-1 in gardenia yellow reached 29.6%, while geniposide in another fraction reached 83.4%. An extract of crocins was obtained from gardenia yellow in a second-stage separation using RP medium-pressure LC, with its color value to be 756 and the content of crocin-1 reaching 60.8%. The separation process was highly efficient, low cost, and compact, which may be informative for purifications of other natural products from complex plant extracts. Keywords: Crocin / Gardenia jasminoides / Geniposide / Macroporous resin / RP chromatography DOI 10.1002/jssc.201300601
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Crocins are secondary metabolites abundant both in saffron (the dried stigmas of Crocus sativus L.) and in gardenia (Gardenia jasminoides Ellis) fruits [1–3]. They are oligosaccharide esters of crocetin (8,8 -diapocarotene-8,8 -dioic acid), a special carotenoid derivate (Fig. 1). Due to their unique water-soluble and antioxidant properties, crocins are applied worldwide as a natural food colorant, mainly in colored juice, jelly, candy, and noodles [4–7]. Numerous studies have also demonstrated a variety of pharmacological actions for crocetin and crocins, including protection against cardiovascular diseases [8] and cerebral ischemia atherosclerosis [9], prevention of retinal degeneration [10], and inhibition of tumor cell proliferation [11]. As saffron is highly valued as the world’s most expensive spice [5], it is of great interest to separate crocins alternatively from gardenia fruit (gardenia yellow) to meet the increasing demand from international markets. Correspondence: Dr. Jiangyun Liu, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China E-mail: [email protected] Fax: +86-512-65882089
Abbreviations: BV, bed volume; MAR, macroporous adsorption resin; MPLC, medium-pressure LC; PDA, photodiode array C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Gardenia jasminoides Ellis is widely cultivated in Southeast Asia, especially in the south of China. Gardinia fruit has been used as a traditional Chinese medicine for its homeostatic, antiphlogistic, choleretic, diuretic, hemostatic, and anticancer properties [12]. Besides crocins, geniposide and other iridoid glycosides, and quinic acid derivatives (such as chlorogenic acid) (Fig. 1) are also present and well characterized in gardenia fruit [13–15]. Geniposide (the major one) and other iridoid glycosides contribute to the many functions of gardenia fruits, such as anti-inflammatory [16], immunosuppressive [17] and other bioactivities. Quinic acid derivatives are also well-known antioxidants beneficial for human health [14]. On the other hand, gardenia yellow is liable to fade and turns green in food application because of the coexistence of geniposide and quinic acid derivatives [18]. Therefore, it is critical to establish an efficient extracting process in industrial practice to separate both gardenia yellow (with high color value) and geniposide simultaneously for integrated utilization of this valuable herb. Extraction methods have been developed extensively for the separation of gardenia yellow, including multiple solvent extraction, membrane filtration, adsorption chromatography ∗ These authors contributed equally to this work. Additional corresponding author Professor Lili Hao, E-mail: [email protected]. Colour Online: See the article online to view the Fig. 3 in colour.
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
315
Figure 1. Chemical structures of crocins, geniposide, and chlorogenic acid.
methods, and imprinted polymer SPE at different process stages [19–21]. The conventional methods for the separation of crocins were performed by means of solid–liquid extraction, liquid–liquid extraction, followed by multistep silica gel column chromatography and semi-preparative HPLC [1,2,4]. However, these methods are not suitable for large-scale production due to the need of bulk amounts of organic solvents and laborious work. In recent years, macroporous adsorption resins (MARs) have been successfully applied for the enrichment of target compounds from crude extracts in industrial practices due to their high efficiency, easy regeneration, and environmentally friendly features [22–29]. Enrichments of gardenia yellow using macroporous resins have been reported, with its color value to be about 300 [23]. However, it remains difficult to separate crocins from gardenia fruits with high purity, high recovery rate, and low cost. Multiplestage separation methods, such as RP silica [24,25], polyamide [26,27], high-speed counter-current [28] chromatography, and crystallization techniques [29] are necessary to be applied in combination with macroporous resin to obtain pure ingredients that are frequently demanded in pharmaceutical applications. These methods could be applied in the further separation of crosins, which remain little reported. It is always important to analyze both target ingredients and impurities at first before a suitable preparation process can be rationally developed. An on-line HPLC with photodiode array (PDA) system has been applied as a powerful method to monitor complex ingredients at multiple wavelengths, as evidenced for HPLC analysis of gardenia fruits [13–15] as well as other traditional Chinese medicines in recent publications [30, 31]. In the present study, a simple and efficient process was developed to enrich both crocins (with color value >700) and geniposide fractions simultaneously from gardenia fruits using macroporous resins and RP medium-pressure LC (MPLC). To fully evaluate the properties of the gardenia extracts at different separation stages, an HPLC–PDA method was applied for the qualitative and quantitative analysis of crocin-1, geniposide, and quinic acid derivates at the same time. Moreover, the color value and optical density ratio value of the crocin extracts were also C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
evaluated. The separation procedure developed here could be informative for purifications of other natural products from complex plant extracts.
2 Materials and methods 2.1 Chemicals, reagents, and samples A crocin-1 standard was purified and identified by ESIMS, NMR spectroscopy, and UV adsorption spectroscopy. Geniposide was purchased from the National Institutes for Food and Drug Control. The purities of crocin-1 and geniposide were 98.7 and 99.2%, respectively, as determined by HPLC. Appropriate amounts of standards were dissolved in methanol to yield the stock solutions at the concentrations of 0.8204 mg/mL for crocin-1 and 1.033 mg/mL for geniposide. Ethanol (analytical grade), methanol (HPLC grade) and other reagents (analytical grade) were all purchased from Shanghai Chemical Reagents (Shanghai, China). Distilled water was purified by a DW100 purification system from Shanghai HiTech Instruments (Shanghai, China).
2.2 Adsorbents MARs including D101, X-5, LX60, AB-8, LX38, LX28, LX8, and LX17 were purchased from Xi’an Sunresin Technology (Shaanxi province, China). Before the adsorption experiments, the resins were weighed and pretreated by soaking in ethanol overnight, subsequently washing once by 4% HCl, distilled water, and 4% NaOH solution, respectively, and finally washing by distilled water thoroughly to remove the monomers and porogenic agents trapped inside the pores during the synthesis process. The moisture contents of the tested resins were determined by drying the beads (1.0 g in wet weight) at 80⬚C to constant weight for over 24 h. The chemical and physical properties of these MARs are summarized in Table 1. www.jss-journal.com
316
J. Sep. Sci. 2014, 37, 314–322
J. Feng et al.
Table 1. Chemical and physical properties of eight macroporous resins in test
Resin
Structure
Polarity
Specific surface area (m2 /g)
Pore size (nm)
Particle size (mm)
Moisture content (%)
D101 X-5 LX60 AB-8 LX38 LX28 LX8 LX17
Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Acrylate
Non Non Non Weak Weak Middle Polar Middle
650–700 500–600 850 480–520 600 500 – ≥460
8.5–9 7.0 7.0 13–14 5.0 7.1 – 13–14
0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25 0.315–1.25
64.8 65.1 72.6 44.5 68.9 59.4 80.2 59.7
2.3 Preparation of the crude extracts of gardenia fruits The dried fruits of Gardenia jasminoides Ellis were collected in Luzhai county, Guangxi province of China in October 2011, and were authenticated by Professor Lili Hao of Soochow University, and a voucher specimen (No. GJ20111001) was deposited there. Gardenia fruits (1.0 kg) were ground to powder, extracted with 40% aqueous ethanol (8 L) under reflux for 1.5 h, repeated twice. The extracting liquors were combined and concentrated to 2.0 L to get the crude extract solution (GJE, with the ratio of raw material to extract volume to be of 0.5 g/mL), which contained 4.120 mg/mL of crocin-1 and 18.28 mg/mL of geniposide. The sample solutions were stored at 4⬚C until use. Deionized water was added to get solutions of different concentrations.
2.4 HPLC analysis of crocin-1 and geniposide Quantitative analysis was carried out by HPLC on a Shimadzu Prominence LC-20A liquid chromatographic system (Shimadzu instruments company, Japan) composed of binary pumps, a PDA detector, and LC solution software. A Cosmosil C18 column (250 mm × 4.6 mm i.d., 5 m) was used at a column temperature of 25⬚C. The mobile phase was consisted of methanol (A) and water (B, with 0.1% acetic acid) with the following gradient program: 0–4 min, 20% A; 4–40 min, 20–90% A. The flow rate was set at 1.00 mL/min, and PDA on-line monitor wavelengths were set at 238 and 440 nm. The retention times of geniposide and crocin-1 were determined to be 15.63 (monitored at 238 nm) and 27.58 min (monitored at 440 nm), respectively. The chromatographic peaks were identified by comparing their respective retention times and UV spectra with those of reference standards, which were eluted in parallel under the same conditions. All samples were filtered through a 0.45 m membrane filter before use. The working calibration curves showed good linearity over the range of 0.8204–11.48 g for crocin-1 and 2.066– 20.66 g for geniposide. The regression curves for crocin-1 and geniposide were Y = (5.5303 × 106 ) X – 1.0221 × 106 (R2 = 0.9993, n = 6), and Y = (1.0976 × 106 ) X – 0.0461× 106 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(R2 = 0.9997, n = 6), respectively, where Y is the peak area of the analyte and X is the injected quantity of crocin-1 and geniposide (g).
2.5 UV/Vis measurement of the color value of gardenia yellow The color value of gardenia yellow was measured as reported [18]. Briefly, dried powder sample (0.15 g) was dissolved in a 100 mL volumetric flask using water as the solvent. Ten milliliters of this sample solution was then transferred to another 100 mL volumetric flask and diluted. Finally, the absorbance of the diluted solution was measured in a standard 1.0 cm pathlength cuvette by a Shimadzu UV-2600 UV/Vis spectrophotometer (Shimadzu, Japan) at 238 (max of geniposide), 328 (max of quinic acid derivates), and 440 nm (max of crocins) (absorbance should be controlled between 0.3 and 0.7). The color value (E) and optical density ratio values (OD) of gardenia yellow were calculated as follows: E = A440 /C
(1)
OD1 = A238 /A440
(2)
OD2 = A328 /A440
(3)
where E stands for the colority of gardenia yellow (A440 with sample amount of 1.0% g/mL using a standard 1.0 cm pathlength cuvette), C is the concentration of the diluted sample solution (g/100 mL), A440 , A238 , and A328 is the absorbance at 440, 238, and 328 nm, respectively.
2.6 Procedures for the batch static absorption and desorption tests 2.6.1 Static adsorption resin screening All macroporous resins were screened through static adsorption tests, which were performed as follows: The hydrated www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
resins (1.0 g dry mass) were put into an Erlenmeyer flask and 50 mL of sample solutions (0.6-fold dilution of GJE, with concentrations of 2.472 mg/mL crocin-1 and 10.97 mg/mL geniposide) were added. The flasks were then shaken (120 rpm) for 5 h at 25⬚C to reach adsorption equilibrium. The solutions after absorption were analyzed by HPLC. After reaching absorption equilibrium, the resins were first washed by deionized water and then desorbed with 50 mL of 95% ethanol. The flasks were shaken (120 rpm) for 5 h at 25⬚C. The desorbed solutions were then analyzed by HPLC. Each experiment was done in triplicate. The preliminary choices of the resins were evaluated by their capacities of static absorption/desorption and desorption ratios. The following equations were used to quantify the absorption and desorption capacities [24]. Absorption capacity: Qe = (C0 − Ce )Vi /W
(4)
where Qe represents the absorption capacity at absorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of solutes in the solutions, respectively (mg/mL); Vi is the volume of the initial feed solution (mL); and W is the weight of the dried adsorbent (g). Desorption ratio: D = Cd Vd /(C0 − Ce )Vi × 100%
(5)
where D is the desorption ratio (%); Cd is the concentration of the solutes in the effluent (mg/mL); Vd is the volume of the effluent.
2.6.2 Adsorption kinetics on LX60 resin The adsorption kinetics curves of crocin-1 and geniposide on the initially selected LX60 resins were studied according to the following process: adding appropriate pretreated resins (equal to 1.0 g dry mass) and 50 mL of sample solutions (0.6fold dilution of GJE) to each flask with a lid, and then shaking for 5 h at 25⬚C (120 rpm). The concentrations of crocin-1 and geniposide in the adsorption process were determined by HPLC at certain time intervals after pretest (5, 10, 20, 30, 45, 60, 90, 120, 180, 240, and 300 min).
2.6.3 Adsorption isotherms on LX60 resin Stock solutions were diluted to get nine different sample solutions in sequence, with concentrations between 0.2472– 4.120 mg/mL for crocin-1 and 1.097–18.28 mg/mL for geniposide. Each sample solution (50 mL) was combined with pretreated resins (equal to 1.0 g dry mass) in shakers and shaken (120 rpm) for 5 h at 25⬚C. The initial and equilibrium concentrations were determined by HPLC. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
317
2.7 Dynamic adsorption/desorption experiments 2.7.1 Dynamic adsorption/desorption on selected resins The separation properties of three selected resins (D101, LX60, and LX17) were further evaluated by the dynamic adsorption and desorption tests. The experiments were conducted in a glass column (30 × 1.5 cm id) packed with 5.5 g (dry mass) selected resins. The bed volume (BV) of the wet-packed resin was 8 mL. Sample solutions (40 mL) flowed through the column at 0.5 mL/min, and the concentration of crocin-1 was 3.296 mg/mL and geniposide was 14.62 mg/mL (0.8-fold dilution of GJE). For the separation process of the three selected resins, the adsorbents were conditioned by gradient elution with water, 20%, and 70% ethanol (each for 48 mL), respectively after adsorption equilibrium of sample solutions at the flow rate of 0.5 mL/min. Each part of the desorption solution was analyzed by HPLC and UV/Vis spectroscopy methods. Each experiment was conducted in triplicate.
2.7.2 Dynamic adsorption/desorption on LX60 resin Several critical parameters, including the concentration and volume of feed solution, the percentage of ethanol in solutions and the volume of the eluents for desorption process were systematically investigated at room temperature. The tests were conducted on a glass column (30 × 1.0 cm id) wetpacked with LX60 (equal to 2.75 g dry mass, 4 mL/BV). The adsorption process was performed by loading feed solution into the pretreated glass column. After adsorption equilibrium for more than 180 min, desorption was completed by gradient elution with solutions of water and ethanol at different ratios. To chose appreciate feed concentration and volume, four different feeding concentrations (0.4-, 0.6-, 0.8-, and 1.0-fold dilution of GJE) of sample solutions were tested. Sample solutions were fed to overload (180, 120, 100, 60 mL, respectively). The eluent fractions were collected every 4 mL and then measured by HPLC. To test the percentage of ethanol in solutions, sample solutions (0.8-fold dilution of GJE, 40 mL) were applied. After the adsorption equilibrium, the adsorbate-laden columns were washed with deionized water, 5, 10, 20, 30, 40, 50, 70, and 95% ethanol (each 24 mL) successively. Each part of eluents was combined and then analyzed by HPLC. Sample solutions (0.8-fold dilution of GJE, 40 mL) were also applied in another test to select the eluent volume needed. After the adsorption equilibrium, the adsorbateladen columns were washed with deionized water (24 mL), 20% ethanol (32 mL), 70% ethanol (36 mL), and 100% ethanol (20 mL) successively. The eluent fractions of 20 and 70% ethanol parts were collected every 4 mL and then measured by HPLC. www.jss-journal.com
318
J. Sep. Sci. 2014, 37, 314–322
J. Feng et al.
2.7.3 Scale-up enrichment of crocins and geniposide from gardenia fruits Scale-up separation was conducted by about 36-fold as that of lab levels. A glass column (45 × 3.5 cm id) was slurry packed with LX60 resin (100 g, dry weight, 150 mL/BV). Sample solution (1.5 L, corresponding to extract from 600 g gardenia fruits) of crude extract (with concentrations of 3.255 mg/mL crocin-1 and 13.83 mg/mL of geniposide) was applied to the column. After sample loading and adsorption equilibrium for more than 180 min, desorption was performed successively with water, 20% ethanol, 70% ethanol (each 900 mL) at a flow rate of 450 mL/h. The effluents of 20 and 70% ethanol parts were collected and dried to yield GJ-1 (geniposide fraction, 20.7 g) and GJ-2 (gardenia yellow fraction, 14.5 g), respectively. Sample fractions were also subjected to analytical HPLC and UV/Vis spectroscopy to determine the contents of geniposide and crocin-1 and the color values. 2.8 MPLC separation of crocins 2.8.1 Purification of crocin and geniposide compounds Separation chromatography was carried out according to Ref. [18], using a MPLC instrument with a PDA detector (BP-Purifier-100, Lisure science corporation, Suzhou, China). The column (15 × 2.5 cm id) was packed with silica gel (300– 400 mesh, Qingdao Haiyang chemical corporation, Qingdao, China) or Cosmosil C18 silica gel (75 m, Nacalai tesque corporation, Tokyo, Japan) by a simple dry-pack method. The BV was 70 mL. As for silica gel column purification, GJ-2 (1.0 g) was dissolved in methanol (10 mL), mixed with 2 g of silica gel and condensed. The dry sample was loaded on the top of the column and eluted with EtOAc/MeOH/H2 O (10:2:1, 900 mL, fraction 1–18) followed by EtOAc/MeOH/H2 O (10:3:1, 800 mL, fraction 19–32). The flow rate was set at 50 mL/min, UV/Vis detection was set at 328 and 440 nm, each fraction was collected at 50 mL, and all the chromatographic experiments were conducted at ambient temperature. The fractions corresponding to crocin-1 (78 mg, fraction 26– 29) were collected and subjected to HPLC to determine the content and the product recovery rate. As for RP separation, C18 column was washed with 100% methanol extensively before use, and then pre-equilibrated with 43% methanol. GJ-2 (1.0 g) was dissolved in 43% methanol (10 mL) and loaded into the column by an injection syringe, eluted with 43% methanol (1.5 L, fraction 1–30), followed by 60% (150 mL, fraction 31–33), 70% (100 mL, fraction 34–35), and 100% (150 mL, fraction 36–38) methanol. The flow rate was set at 50 mL/min, UV detection was set at 328 and 440 nm, and all the chromatographic experiments were conducted at ambient temperature. The peaks corresponding to quinic acid derivatives or crocins were collected and subjected to analytical HPLC to determine the content and the C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
product recovery rate, respectively. Trace amount of geniposide was detected in fractions 3 and 4. Quinic acid derivatives were detected in fractions 5–22. Crocin-1 (164 mg), crocin-2 (19 mg), and crocin-3 (51 mg) were obtained from fractions 24–28, 32–33, and 35, respectively, and identified by comparison of their NMR and UV spectroscopic data with published data [1, 2, 32]. GJ-1 (1.0 g) was dissolved in hot EtOAc (20 mL) and filtered, cooled to room temperature and then filtered again to give geniposide (460 mg). Geniposide was identified by comparisons of its retention time in HPLC and its UV spectrum with a reference standard. 2.8.2 Preparative separation of crocins by RP MPLC For the preparative separation of crocins, an RP column (25 × 3.5 cm id) packed with Cosmosil C18 silica gel (75 m) was applied. The BV was 240 mL. The column was pre-equilibrated with 40% methanol. GJ-2 (10.0 g) was dissolved in 40% methanol (100 mL) and loaded into the column by an injection syringe, eluted with 40% methanol (3.0 L) to remove impurities, followed by methanol (1.0 L). The flow rate was set at 100 mL/min, UV detection was set at 328 and 440 nm, and all the chromatographic experiments were conducted at ambient temperature. The peaks corresponding to crocins (GJ-3, 4.76 g) or quinic acid derivatives (GJ-4, 3.41 g) were collected and subjected to analytical HPLC to determine the content and the product recovery rate, respectively.
2.9 Statistical analysis Data were expressed as means ± standard error of three separate experiments, and a t-test was used for determining the statistically significant differences (P < 0.05).
3 Results and discussion 3.1 The preliminary choice of the resins 3.1.1 Batch static adsorption/desorption experiments Adsorption characteristics of macroporous resins are not only closely related to polarity and size of adsorbates, but also to dimensional structure (specific surface area, pore diameter, and pore volume) of adsorbents, and to the adsorption medium. Eight typical macroporous resins (Table 1) were therefore selected and tested for their adsorbing and separation performance. The results of static adsorption tests and desorption are shown in Table 2. MARs in this survey demonstrated different adsorption capacities, ranging from 34.3–95.4 mg/g for crocin-1 and 75.9–186.8 mg/g for geniposide. By comparison of different polystyrene resins, three nonpolar resins www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
319
Table 2. Adsorption capacities, desorption capacities, and desorption ratios of eight resins for crocin-1 and geniposide
Resin
D101 X-5 LX60 AB-8 LX38 LX28 LX8 LX17
Adsorption (mg/g)
Desorption (mg/g)
Crocin-1
Geniposide
92.1 ± 90.2 ± 95.4 ± 56.1 ± 58.9 ± 58.2 ± 34.3 ± 89.4 ±
134.2 152.5 186.8 120.7 99.7 75.9 89.8 161.6
1.9 4.1 3.6 1.2 3.5 2.9 1.7 3.8
± ± ± ± ± ± ± ±
2.7 3.2 3.1 1.3 1.8 0.9 2.1 3.6
Desorption ratio (%)
Crocin-1
Geniposide
78.4 ± 75.3 ± 88.7 ± 48.2 ± 53.8 ± 46.8 ± 25.5 ± 75.8 ±
106.2 133.0 168.1 111.3 84.8 56.9 80.5 138.6
2.5 3.2 3.1 3.9 4.1 1.9 0.8 2.7
(D101, X5, and LX60) showed apparent higher adsorption capacities than other weak-polar/polar resins, indicating a preferred hydrophobic interaction with the adsorbates (aglycone groups). LX60 achieved a better adsorption/desorption capacity than D101 and X5, partly because of its higher specific surface areas than that reported by Yang et al. [22]. LX17 belonging to the polyacrylate class also exhibited excellent adsorption/desorption efficiency similar to that of D101 or X5. This may be attributed to the main electrostatic attraction and hydrogen-bonding interactions between the polyacrylate resins and adsorbates (glucosyl group) [25]. Accordingly, three resins, LX60, D101, and LX17 were selected for further dynamic separation investigations.
3.1.2 Dynamic separation performance of selected resins Dynamic separation properties of three selected resins toward crocin-1 and geniposide were tested as shown in Table 3. Geniposide could be efficiently desorbed by 20% ethanol for three resins, while crocin-1 was not desorbed until 70% ethanol. Two products, geniposide (GJ-1) and gardenia yellow (GJ-2) extracts could thus be obtained in this stage. Generally, the colority of the gardenia yellow product reached 300 after purification by macroporous resin such as HPD722 [23]. The color value of two samples performed better to be 392 (for D101) and 423 (for LX60). This was in agreement with higher adsorption capacities of the resins applied as evidenced in static tests (Table 2). The efficient removal of geniposide using 20% ethanol could also contribute to higher purity of
± ± ± ± ± ± ± ±
1.4 1.6 3.3 2.5 1.1 1.2 2.9 2.1
Crocin-1
Geniposide
85.1 83.6 92.9 85.9 91.2 80.4 74.5 84.7
79.1 87.2 89.9 92.2 85.1 74.8 89.7 85.8
GJ-2. Considering of its higher adsorption capacity and purity of products, LX60 was chosen as optimal resin for the enrichment of crocin-1 and geniposide.
3.2 Static/Dynamic experiments on LX60 resin 3.2.1 Adsorption kinetics on LX60 resin The adsorption kinetics curves of crocin-1 and geniposide on LX60 are shown in Supporting Information Fig. S1A. The adsorption of crocin-1 and geniposide on LX60 resin increased with increasing adsorption time. The adsorption increased rapidly in the first 60 min, and then it increased slowly, and finally reached equilibrium after 180 min for crocin-1 and 120 min for geniposide, indicating that LX60 belongs to the slow adsorption resin type. Therefore, the batch adsorption equilibrium tests were run for over 180 min. Adsorption kinetics parameters for both crocin-1 and geniposide on LX60 were calculated (Supporting Information Table S1). A pseudo-second-order kinetics model was suitable to describe the entire adsorption process on the resin, with calculated correlation coefficient (R2 ) to be 0.9923/0.9986 for both adsorbates. The pseudo-second-order kinetics implied that concentrations of both adsorbates and adsorbents were involved in rate-determining steps during the adsorption process. These results indicated that the adsorption process was controlled by two or more rate-limiting steps such as external diffusion, boundary layer diffusion, and intra-particle diffusion [24].
Table 3. Dynamic adsorption capacities and properties of crocin-1 and geniposide on three selected resins
Resin
D101 LX60 LX17
Adsorption (mg/g)
Content (%)
Crocin-1
Geniposide
Crocin-1
Geniposide
Crocin-1
Geniposide
E
OD1
OD2
49.7 ± 2.3 54.5 ± 1.9 48.4 ± 2.1
121.5 ± 3.1 156.3 ± 3.5 113.5 ± 3.9
23.0 ± 0.7 25.6 ± 0.9 24.5 ± 1.1
79.7 ± 1.6 83.4 ± 1.3 80.8 ± 0.5
80.6 82.4 79.7
73.6 75.7 68.2
392 423 347
0.420 0.407 0.512
0.409 0.385 0.601
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Recovery (%)
Color value
www.jss-journal.com
320
J. Feng et al.
3.2.2 Adsorption isotherms on LX60 resin Equilibrium adsorption isotherms of LX60 resin were measured at 25⬚C as shown in Supporting Information Fig. S1B. The adsorption capacity of LX60 resin toward both crocin-1 and geniposide increased as the adsorbate concentrations increased, and reached saturation at 3.296 mg/mL for crocin-1 and 14.62 mg/mL for geniposide, respectively. The results provided reference values for scaling up the process with regard to crude extract concentrations prior to the chromatography. To simulate the equilibrium adsorption behaviors, Langmuir and Freundlich parameters were calculated and summarized in Supporting Information Table S2. The Langmuir equation described the adsorption behavior of crocin-1 and geniposide on LX60 resin better because the calculated correlation coefficients (0.9924 and 0.9946, respectively) of the Langmuir equations were higher than that of the Freundlich equations. These results suggested the monolayer coverage of crocin-1 and geniposide onto the resin. The theoretical maximum adsorption capacity (Qmax ) of crocin-1 (geniposide) determined from the Langmuir equation was 91.74 mg/g (188.7 mg/g). In the Freundlich equation, the adsorption is very difficult to achieve if 1/n value is above 2 [24]. In Supporting Information Table S2, the 1/n values are 0.2366 and
J. Sep. Sci. 2014, 37, 314–322
0.3215, respectively, which indicates that the LX60 resin is suitable for separating crocin-1 and geniposide.
3.2.3 Dynamic adsorption and desorption tests on LX60 resin 3.2.3.1 Effect of feeding concentration and volume In the process of dynamic adsorption, the effects of feeding concentration and feeding volume on adsorption capacity were investigated, and the results of leakage curve at different concentrations were obtained (Fig. 2A and B). The adsorption capacities slightly increased but fell back with the increment of initial feed concentrations due to competition of active sites of LX60 resins by impurities in the crude extracts and therefore limiting the diffusion of crocin-1 into the micropores of LX60. The breakthrough point generally refers to the circumstance under which the adsorbate concentration in the eluent reaches 5% of that in the sample solution applied. In the present study, the adsorption capacity did not increase apparently when the initial concentration of crocin-1 (geniposide) was >3.296 mg/mL (14.62 mg/mL), and the corresponding feed volume was 40 mL (with ratio of corresponding raw material and resin dry mass to be 5.8 g/g).
Figure 2. Effects of feed concentration (A) and volume (B), and elution solvent effects of ethanol concentration (C) and volume (D) on content of crocin-1 and geniposide.
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 314–322
321
Table 4. Contents and color values of crocin-1 and geniposide in different extracts
Extract
Content (%)
GJE
0.8601a) 3.806b) 83.38b) 29.65a) 60.78a)
GJ-1 GJ-2 GJ-3
Yield (%)
3.45 2.41 1.13
Recovery (%)
75.7 83.1 81.2
E
OD1
OD2
67
1.831
0.517
390 756
0.421 0.148
0.375 0.142
Note: a)Crocin-1; b)Geniposide.
3.3 Scale-up preparation of crocins and geniposide on LX60 resin
Figure 3. HPLC–PDA chromatograms of different fractions of gardenia fruits monitored at 440 (I), 238 (II), and 328 (III) nm simultaneously. (A) Mixture solution of crocin-1 and geniposide reference substances; (B) GJE (crude extract); (C) GJ-1 (geniposide) fraction; (D) GJ-2 (gardinia yellow) fraction; (E) GJ-3 (crocin) fraction; (F) GJ-4 (quinic acid derivates) fraction. 1 geniposide; 2 crocin-1.
3.2.3.2 Effect of the eluent concentration and volume on desorption In the process of dynamic adsorption, the effect of ethanol concentration for the desorption process was investigated. After adsorption equilibrium, the adsorbate-laden column was first washed with deionized water, and then desorbed by gradient elutions with 5–95% ethanol, respectively. Figure 2C showed that geniposide could be desorbed mainly with 5–30% ethanol, and crosin-1 could be desorbed after 20% ethanol. The procedure was thus set by elution with water at first to remove impurities, followed by 20% (to afford geniposide fraction) and 70% (to desorb crocins efficiently) ethanol. Regarding the eluent consumption of the separation process as an important factor, the volume of eluent was investigated under the optimal separation conditions. The concentrations of crocin-1 and geniposide in the eluent were plotted against eluent volumes, and the elution curves were obtained as shown in Fig. 2D. The concentrations of both analytes in the eluent reached maximum at the third fraction, then decreased rapidly, and fell to
