J. Cent. South Univ. Technol. (2007)03-0357-06
DOI: 10.1007/s11771-007-0071-7
Purification of chlorogenic acid in Flos Lonicerae with system of
polar ordered resins
XIANG Zhi-nan(向智男)1, ZHAN Yu(战 宇)2, NING Zheng-xiang(宁正祥)1
(1. College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, China;
2. School of Biological and Chemical Engineering, Guangzhou University, Guangzhou 510091, China)
Abstract: A system of polar ordered resins was established for purification of chlorogenic acid in Flos Lonicerae. It was composed of three reversed phase resins, AB-8, DM-130 and NKA-9, representative for their gradually increased polarity and selectivity. A method of RP-HPLC was used for determination of chlorogenic acid. And the performance of adsorption and desorption for chlorogenic acid with the system of polar ordered resins was studied. Furthermore, the effects of concentration, pH and flow rate of the adsorbate on adsorption ability were researched. It is indicated that the optimum parameters for chlorogenic acid are as follows: pH 3.5 with a flow rate of 2.5 BV/h, the concentration of extract solution at 0.50, 0.40, 0.30 g/L respectively for the adsorptive operation twice, and 6.93, 8.66, 10.39 mol/L ethanol used as gradient eluants. The purity of resulted product of chlorogenic acid arrives 70.20% with yield of 89.79%. With simple procedures, low costs and high purity product, the method of system of polar ordered resins followed by sequential reversed phase separations can be used to refine the chlorogenic acid in the extraction of Flos Lonicerae.
Key words: chlorogenic acid; Flos Lonicerace; polar ordered adsorption and desorption; purification
1 Introduction
Flos Lonicerae (Honeysuckle Flower, JinYinHua in Chinese), derived from the flower buds of several species of the genus Lonicera (Cap-rifoliaceae), is a kind of Chinese traditional medicine herb used in clinic for centuries, such as the treatment of affection by exopathogenic wind-heat or epidemic febrile diseases at the early stage, sores, carbuncles, furuncles and swellings[1-2]. It has medicinal efficiency of clearing away heat and toxic materials, and is used to treat warm diseases, dysentery due to damp-heat pathogen, and carbuncle, etc[3]. Moreover, it can suppress the N-nitrosating reaction and inhibit hepatic glucose 6-phosphatase [4-6], which may be a significant factor in diabetes. The results of chemical study showed that Flos Lonicerae contains chlorogenic acid (a group of quinyl ester of hydroxycinnamic acids abbreviated as CGA), luteolin, lonicerin, inositol, etc. Among them, CGA is considered as the main constituent that has antioxidant activity[7-8]. Because of the certainty of curative effects, CGA is now used not only as one of the quality control in crude herbal drugs, but also in some preparations of Chinese patent medicine and health food, in which the extract of Flos Lonicerae is added[9-10].
A number of conventional methods, such as precipitation, extraction, crystallization and column chromatography, were concerned on the CGA, among which the tiptop purity just reached 36.28%[11-12]. An ideal purity for chlorogenic acid from the crude extract of Flos Lonicerae was achieved by high-speed-counter- current chromatography (HSCCC). However, the enormous cost and the complex operation for this technology should be cognized, besides its low productivity[13].
Meanwhile, many scholars have done some work on the resin adsorption for CGA. LIU and QIU[14] selected NKA-Ⅱ resin as the absorbent to extract and purify chlorogenic acid from leaves of Eucommia ulmoides, with the adsorptive rate of 80.99%. LI et al[15] found NKA-9 resin was the better one for purification of chlorogenic acid when compared with resins X-5, AB-8 and S-8, with the purity of resulted product of 25.12%. MA et al[16] chose the specific macorporous resin XDA-5 to adsorb and isolate chlorogenic acid and flavonoids, and the content of purified chlorogenic acid in solid extract was 36.65%. LI et al[17] introduced D101-column separation-precipitation method for assaying chlorogenic acid in Chinese medicinal preparations, and evaluated that the D101 resin could remove a great portion of impurities.
In this research, an effective system of polar ordered macroporous resins for purification of CGA in the traditional herb Flos Lonicerae was established, and its performance of adsorption as well as the optimal desorption conditions were studied.
2 Experimental
2.1 Materials and reagents
Flos Lonicerae was collected in spring from Zhanjiang National Plantation of Lonicera Confusa DC, Guangdong Province (China). Macroporous adsorptive resins of AB-8, DM-130 and NKA-9 were obtained from Nankai Chemical Plant. Standard sample of CGA was purchased from Medical and Biochemical Authentication Research Institute (China). Methanol and glacial acetic acid were chromatographic grade; water was deionized and the other solvents were all of analytical grade.
2.2 Apparatus and chromatography condition
KQ5200 DE numerical control ultrasonic cleanout equipment(Kunshan, China), ALPHA1-4 freeze dryer (Martin Christ, Germany), TU-1810 spectrophotometer (Beijing, China), UV-1 triple-purpose ultraviolet analyzer (Shanghai, China), RE-52 rotary vacuum evaporator (Shanghai, China), FA2004 electron balance (Shanghai, China), laminating glass chromatography column (inside diameter 3 cm, length 60 cm, Changsha, China), microfiltration membrane (aperture 0.22 μm, Shanghai, China) and silica gel (Qingdao, China) were used.
Chromatographic determination was performed on a HPLC system (Summit, Dionex Co., Germany), which consisted of two HPLC P680 pumps, an ASI-100 automated sample injector, a TCC-100 thermostat column compartment, a PDA-100 photodiode array detector, an UCI-50 universal chromatography interface and an Alltech ELSD 2000ES detector. A reversed-phase symmetry C18 column (inside diameter 4.6 mm, length 200.00 mm, stuffing diameter 10 μm, Waters) at a column temperature of 30 ℃ was used to separate CGA. The mobile phase consisted of two solvent components: 1.0 % aqueous acetic acid and methanol (48?52,volume ratio). The flow-rate was 1.0 mL/min. A sample loop of 10 μL was used for sample injection. The absorbance spectra (250-400 nm) were collected continuously during the course of each analytical run. Qualitative analysis was made by retention time and quantitive analysis by peak area. Evaluation and quantification were both performed on the Millennium chromatography data system (Waters).
2.3 Extraction method
Before experiment, fresh Flos Lonicerae was dried at 50 ℃ until the mass was unchangable. Certain amount of the pulverized samples (particle diameter: 0.45 mm) were soaked with 8-folds of water, and pH value was adjusted at 3.5 with citric acid simultaneously. After 12 h soakage, they were treated with 0.05% cellulase（volume fraction）for 30 min at 50 ℃. Then, the solution was heated in water bath at 70 ℃ for 40 min, of which the filtered residues were extracted by 8-folds of water in water bath at 60 ℃ for 20 min. Both the filtrates were combined and centrifuged to obtain the supernatant. After filtrated via a 0.22 μm microfiltration membrane, the solution was concentrated by rotary evaporation into appropriate degree so as to obtain the crude extraction solution of CGA, which was marked with (Ⅰ) and stored at 4 ℃[18-19].
2.4 Establishment of system of polar ordered resins
As shown in Table 1, the polarity of adsorptive resins AB-8, DM-130 and NKA-9 is gradually stronger and stronger. Through static state adsorptive test, in which the content of CGA was determined by ultraviolet spectrophotometry, the properties of adsorptive capacity (Q) and adsorptive rate (E) for each resin were worked out:
(1)
(2)
where c0 represents the concentration of adsorbate solution (g/L); c1 represents the concentration of the solution after adsorption (g/L); V0 represents the volume of adsorbate solution (mL); V1 represents the volume of the solution after adsorption (mL) and m represents the mass of resin (g).Moreover, the experiment of desorption for resins was done to select the appropriate concentration of ethanol solution as the eluant. These resins were installed in the columns respectively by wet method. Then the CGA extraction solution was pumped in the resin AB-8 column at the regulated flow rate until 7-folds was finished, and this operation was repeated twice. Afterwards, the ethanol solution in appropriate concentration was used to elute the resin until there was no specific blue-purple fluorescence when being detected by ultraviolet analyzer under 365 nm. The resultant solution was concentrated by rotary vacuum evaporation and dried by freeze drier into powder, which marked with (Ⅱ). Whereafter, the powder (Ⅱ) was dissolved in water, of which the dissolution would be acted as the sample solution to resin DM-130 column. Similarly, the purified powder for CGA by DM-130 was marked with (Ⅲ). After the next purification process of resin NKA-9, the powder (Ⅳ) was obtained, which was just the
Table 1 Properties of macroporous resins in experiment for chlorogenic acid[20-22]
resultant purified CGA by this developed measure of the system of polar ordered resins.
2.5 Anion exchange resin for CGA purification
CGA is the phenolic acid, and under UV spectra scanning its maximal adsorptive wavelength is 327 nm. Theoretically it would exist as the acid radical anion at the modified pH value, while other impurities, especially the compound of iridoid has the maximal adsorptive wavelength at 238 nm[23]. Then the purified CGA would be gained after the adsorption and separation by alkali anion exchange resin.
The processes of installing resins and adsorption were just similar to the method of the system of polar ordered resins. Afterwards, the solutions of HCl, H2SO4, citric acid, salicylic acid, ammonia and sodium hydroxide (NaOH) were respectively used to elute the resin. The UV spectrum of the outflow was scanned followed by the absorbency test.
3 Results and discussion
3.1 Properties of resins
Three macroporous resins of AB-8, DM-130 and NKA-9 were representative for the polarity. The properties of the resins for CGA solutions (10.0 g/L, 10 mL), such as adsorptive capacity, adsorptive rate and the purity of its resultant product, were examined by the static state adsorption and desorption experiments.
From the data listed in Table 1, the adsorptive capacity of resin of AB-8 (low polarity), DM-130 (semi polarity) or NKA-9 (strong polarity) solely, gradually descends along with increasing polarity. However, NKA-9 has the easiest desorption, best selectivity and highest purity, while AB-8 has the largest surface area and the highest adsorptive rate except for its lowest selectivity among three resins.
3.2 Adsorption properties of system of polar ordered resins for CGA
In most cases, low concentration of the prepared solution is propitious to the adsorption of macroporous resins. It is suggested in single factor experiment that the optimal concentrations of the prepared solution are 0.50, 0.40 and 0.30 g/L for AB-8, DM-130 and NKA-9, respectively (Fig.1(a)).
CGA can exist as molecule when being set in weak acidity so that its hydrophobicity is stronger and easier to be adsorbed, whereas the type of lactone in strong acidity or alkali surroundings it can be hydrolyzed easily. So CGA of ionic species, when being hydrolyzed in alkali, is difficult to be adsorbed. The test data illustrate that the appropriate pH value is 3.5 (Fig.1(b)).
The flow rate is another important factor for the rapidness can bring on the decrease of adsorptive quantity with the low efficiency at the undersize flow-rate. It is shown that a rate of 2.5 BV/h makes good effect when CGA extract passes this resin system at pH=3.5 (Fig.1(c)).
3.3 Desorption properties of system of polar ordered resins for CGA
The ethanol with different concentrations was used twice separately as the eluant for the three resins. At the same time, the content of CGA was determined by UV spectrophotometry. The results show that, the high desorption quotiety conditions are as follows: ethanol with the concentration of 6.93, 8.66 and 10.39 mol/L as
Fig.1 Effect of prepared solution of chlorogenic acid on adsorption ability of system of polar ordered resins
(a) Concentration; (b) pH value; (c) Flow rate
the optimal eluants, and the desorptive rate at 95.50%, 98.32% and 94.37% for the corresponding resins of AB-8, DM-130 and NKA-9(Fig.2).
3.4 Quantitative analysis by reversed phase-high performance liquid chromatography (RP-HPLC)
3.4.1 Calibration curves
Stock solution of CGA was prepared by dissolving standard substance CGA in 50% aqueous methanol and diluted to appropriate concentration ranges: 0.175 5, 0.351, 0.702, 1.404, 2.808, 4.212 and 5.616 mg/mL. Accurately 10.0 μL of each of the prepared solution was
Fig.2 Effect of concentration of ethanol solution on desorption of system of polar ordered resins
injected in RP-HPLC by the automated sample injector individually. The calibration curve was performed with these seven different concentrations in triplicate by plotting the peak area versus concentration. The chromatograms and data were recorded (Fig.3(a)), and the linear equation of y＝22.473x+1.25×10-3 (y: peak area (mAU), x: sample concentration (mg/mL), r＝ 0.999 3) was gained by regression analysis. The results demonstrate that the standard sample of CGA has a good linear relation within the range of 1.755-56.160 mg/mL.
3.4.2 Determination by RP-HPLC
The flower buds of Lonicera Confusa DC were collected and dried until constant mass was got at 50 ℃. Approximately 2.5 g flower buds were pulverized, and the mixed powders (Ⅱ), (Ⅲ) and (Ⅳ) were all accurately weighed and dissolved with methanol into a 25 mL flask separately. They were then filtered through No.1 Xinhua filter paper (Hangzhou Paper Factory, China) and 1 mL of the succeeding filtrate was transferred to a 2 mL volumetric flask and filled with methanol to the mark. The resultant solution was filtered through a 0.45 μm syringe membrane filter (Type Millex-HA, Millipore, USA) prior to RP-HPLC. The contents of the analytes were determined from the corresponding calibration curve, and the results are listed in Table 2.
The HPLC spectrum of powder (Ⅳ), which is namely the resultant purified product of the system of polar ordered resins, is shown in Fig.3(b). And it is suggested that the spectrum of the resultant purified chlorogenic acid is basically the same as that of the standard sample.
3.5 Stability and precision of system of polar ordered
resins
Five samples with equal volume of 20 μL, which were under the same optimized extraction condition and
Table 2 Content of chlorogenic acid in each purified procedure product of system of polar ordered resins
(Ⅰ): Freeze-dried powder from crude extraction solution of CGA from Flos Lonicerae; (Ⅱ): Freeze-dried powder of product of CGA after purification procedure of resin AB-8; (Ⅲ): Freeze-dried powder of product of CGA after purification procedure of resin DM-130; (Ⅳ): Freeze-dried powder of resultant product of CGA purified by system of polar ordered resins
Fig.3 Chromatograms of chlorogenic acid by RP-HPLC analysis
(a) Standard sample of chlorogenic acid; (b) Purified product of chlorogenic acid through system of polar ordered resins
purified by the system of polar ordered resins, were individually obtained every certain period of time in 24 h. The filtrate was analyzed by RP-HPLC. By comparing these evaluated data, it is illustrated that the purification system of polar ordered resins has good stability in 24 h.
Furthermore, variations of the content of CGA in five samples with the equal volume, which were the resultants of the same processing as above, were chosen to determine the precision of the developed measure, analyzed by RP-HPLC. The result with the value 0.12% of RSD suggests that this purification system has a satisfied precision.
3.6 Recovery and reproducibility of system of polar ordered resins
The determination of the recovery of CGA was carried out by standard addition method with purification under the same optimized conditions for 5 times, as listed in Table 3. The obtained recovery of CGA is 89.79%.
Table 3 Recovery of chlorogenic acid for system of polar ordered resins
A: Chlorogenic acid quantity of powder sample determined by RP-HPLC; B: Amount of added chlorogenic acid as standard; C: Amount of chlorogenic acid determined in sample with added standard after purification by system of polar ordered resins; RSD: Relative standard deviation
To study the reproducibility of the purification of the system of polar ordered resins for CGA, six samples were processed under the optimum condition and determined by RP-HPLC. As indicated in Table 4, this purification system has a good repeatability.
3.7 Effect of purification for CGA by anion exchange
resin
When using the method of gradient or constant elution, the absorbency of outflow is always A327238, and the content of CGA is trace. While the impurities, such as the compound of iridoid, have not been removed. The results illustrate that the method of anion exchange resin cannot purify the CGA product as simply and conveniently as the measures of system of polar ordered resins.
Table 4 Reproducibility of chlorogenic acid for system of polar ordered resins
4 Conclusions
1) An approach of the purification system of polar ordered resins consisting of macroporous resins AB-8, DM-130 and NKA-9 that are representative for their gradually increasing polarity and selectivity for CGA is propsed. And the sequential purification of CGA by a series of these three reversed phase separations is used to refine the extraction of the herb Flos Lonicerae.
2) The performance of adsorption and desorption for chlorogenic acid with the system of three polar ordered resins is studied and the optimum conditions are as follows: a flow rate of 2.5 BV/h at pH 3.5, the concentration of the adsorbate solution at 0.50, 0.40, 0.30g/L, and ethanol of 6.93, 8.66 and 10.39 mol/L used as gradient eluant for the ordered resin AB-8, DM-130 and NKA-9, respectively.
3) The way of thin-layer chromatography and RP-HPLC is used to carry through the qualitative and quantitative analysis for CGA. After purification by the system, the purity of CGA can achieve 70.20％, accompanied with recovery of 89.79％.
4) Compared with anion exchange chromatography followed by reversed phase separation, the measure of system of polar ordered resins is demonstrated to be more efficient and convenient in practical operation.
5) This way of the system of polar ordered resins is recommended to make the product of CGA attain higher purity, and is supposed to make sweeping industrialization.
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(Edited by YANG Bing)
Foundation item: Project(2005A20303002) supported by the Science and Technology Plan Item of Guangdong Province, China; Project (2006J1-C0251) supported by the Science and Technology Bureau Foundation of Guangzhou, China
Received date: 2006-06-28; Accepted date: 2006-08-27
Corresponding author: XIANG Zhi-nan, PhD; Tel: +86-13533845658; E-mail: xiangwanzi2003@163.com
