Objective To study the extraction and purification of chlorogenic acid from Eucommia ulmoides leaves. Methods The purified product was extracted by water extraction, flocculation and decoloration, resin adsorption and recrystallization. Results The concentrated aqueous extract of Eucommia ulmoides leaves was flocculated with 1% chitosan to remove impurities, and decolorized with activated carbon to obtain injection. The injection was adsorbed with NKA-9 resin, desorbed with 50% ethanol, and the concentrated crude eluent was recrystallized with methanol to obtain chlorogenic acid with purity ≥97% and extraction rate ≥65%. Conclusion The extraction and purification methods of chlorogenic acid from Eucommia ulmoides leaves were optimized and improved, which provided a reference for the comprehensive utilization and industrial development of Eucommia ulmoides resources.
Key words: chlorogenic acid; Excerpts; Purification; Eucommia leaves
Study on extraction and purification of chlorogenic acid from Eucommia ulmoides Oliv. leave
Yuan Hua, Deng Liang, Yang Xiao? Jun, Yan Zhi? Guo, Wu Yuan? hard
(School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073)
Objective To study the best extraction and purification technology of chlorogenic acid from Eucommia ulmoides Oliv. Leaves. Methods water extraction, 1% chitosan flocculation, activated carbon decolorization, NKA? 9 resin adsorption, 50% ethanol desorption and methanol recrystallization. Results The extraction rate of chlorogenic acid was 68% and the purity was 97%. Conclusion The extraction and purification process of chlorogenic acid from Eucommia ulmoides Oliv. was optimized and improved. The leaves were investigated. This is helpful to the industrial production of chlorogenic acid.
Key words: chlorogenic acid; Extract; Purification; Eucommia ulmoides Oliv
Eucommia ulmoides Oliv It is a unique economic tree species in China, and its medicinal value has always been concerned. Chlorogenic acid is the main effective component of Eucommia ulmoides Oliv. It has antibacterial, antiviral, cholagogic, hepatoprotective, antihypertensive and free radical scavenging effects. More than 2000 years ago, it was listed as the top grade of traditional Chinese medicine in Shennong Herbal Classic. Recent studies show that the main chemical components of Eucommia ulmoides leaves are basically the same as Eucommia ulmoides bark, and the chlorogenic acid content in Eucommia ulmoides leaves is richer, reaching 1%~5% of the dry leaf weight, so the extraction of chlorogenic acid from Eucommia ulmoides leaves with abundant raw materials and low price has become a research hotspot [1 ~ 3]. Among them, the extraction and purification method of macroporous adsorption resin proposed by Hua Qian [4] and Liu Junhai [5] shows a certain industrial prospect, but the reported resin separation effect is inconsistent, and there is no report on the purity and yield of the final product. Besides chlorogenic acid, Eucommia ulmoides leaves extract also contains tannins, protein, polysaccharides and other substances. In order to obtain high purity chlorogenic acid, it is necessary to separate and purify the extract in multiple steps. On the basis of literature work, this paper improved the extraction and preparation process around the industrialization goal, and extracted and prepared chlorogenic acid with high purity and high yield through water extraction, flocculation decoloration, resin adsorption and recrystallization.
1 materials and methods
1. 1 materials and instruments Eucommia ulmoides leaves were collected from Wufeng, Hubei (air-dried, crushed); The main reagents such as ethanol, methanol and dilute hydrochloric acid are analytically pure; Chitosan is a biochemical reagent; NKA？ 9、AB？ 8、NKA？ Type Ⅱ resin (Nankai University Chemical Plant); Chlorogenic acid (China Institute for the Control of Pharmaceutical and Biological Products). ZF？ Type I uv analyzer (Shanghai Gucun photoelectric instrument factory); Agilent 1 100 high performance liquid chromatograph.
1.2 extraction and purification process Accurately weigh Eucommia ulmoides leaves 10.0 g, dynamically extract with 2× 120 ml deionized water at 80℃ 150 min, filter, concentrate the extract under reduced pressure to 100 ml, and add 4ml/kloc-to the concentrated solution. 9. Absorb the sample supernatant with resin, elute with 50% ethanol solution, concentrate the eluate under reduced pressure to obtain crude chlorogenic acid, and recrystallize with methanol to obtain pure chlorogenic acid. The extraction rate is ≥65% (calculated by chlorogenic acid content in Eucommia ulmoides leaves) and the purity is ≥97%.
1.3 determination of chlorogenic acid content the determination of chlorogenic acid content was carried out according to references [6, 7].
Two results
2. 1 extraction conditions
2. 1. 1 Selection of extraction solvent Chlorogenic acid in Eucommia ulmoides leaves is generally extracted with 40% ethanol aqueous solution. Using ethanol aqueous solution to extract not only increases the cost of solvent, but also increases the fat-soluble components in the extract, which brings difficulties to the subsequent treatment. Chlorogenic acid has high solubility in hot water. In this paper, water is selected as the extraction solvent.
2. 1.2 Selection of extraction temperature With the increase of extraction temperature, the yield of chlorogenic acid increased continuously, and when the temperature was higher than 80℃, the yield of chlorogenic acid began to decrease. With the increase of temperature, the dissolution of other components in Eucommia ulmoides leaves is also increasing. Considering the thermal instability of chlorogenic acid, the extraction temperature is controlled below 80℃.
2. The influence of1.3 extraction time. With the extension of time, the yield of chlorogenic acid gradually increased, especially within 50 ~ 150 min, and the two showed a good linear relationship, and then gradually reached an extraction equilibrium state.
2. 1.4 Effect of pH value of extraction solution Under the same conditions of temperature, solid-liquid ratio and extraction time, dilute hydrochloric acid was used to adjust the pH value of extraction solvent, and when the pH value was 4, the yield of chlorogenic acid was higher. Therefore, the best extraction conditions of chlorogenic acid are: Eucommia ulmoides leaves per piece. Yes 10.0 g, dynamically extracted with 2× 120 ml deionized water at pH=4 and temperature 80℃ 150 min.
2.2 Flocculation Decolorization In the extraction process of traditional Chinese medicine, the method of water extraction and alcohol precipitation is often used, with the purpose of removing invalid components such as starch, colloid, protein, mucus, pectin, polysaccharide, inorganic salt, etc. It was found that the higher the alcohol concentration, the better the impurity removal effect, but the loss of effective components caused by alcohol precipitation also increased. In addition, the process of water extraction and alcohol precipitation still has some shortcomings, such as large amount of ethanol, high production cost and low safety factor, so it is necessary to improve the process. Flocculation clarification method is to add flocculant to the extract of traditional Chinese medicine or concentrated extract, and remove some substances in the solution through adsorption bridging and electric neutralization, so as to achieve the purpose of separation and purification [8]. Through the selection and comparison of flocculant types, concentration, flocculation temperature and acidity, the optimum flocculation and decoloration process was determined as follows: adding 4ml 1% chitosan solution to each 100 ml concentrated solution, stirring, standing at 60℃ for 60 min, filtering, and decoloring the filtrate with 1g activated carbon to obtain a clear injection.
2.3 Selection of Macroporous Adsorption Resins Combined with the characteristics of Eucommia ulmoides leaves extract, three polar macroporous adsorption resins NKA-9, AB-8 and NKA-II were comparatively selected in this paper. After pretreatment and regeneration, static adsorption and desorption experiments were carried out. See table 1 for the adsorption capacity, adsorption rate and desorption rate of chlorogenic acid solution (4.8 mg ml-1,10 mL) by three different types of macroporous adsorption resins.
Table 1 adsorption and desorption of chlorogenic acid by three resins (omitted)
From the table 1, it can be seen that the adsorption capacity and adsorption rate of chlorogenic acid by three kinds of macroporous adsorption resins are high, but chlorogenic acid adsorbed by AB-8 resin is difficult to desorb and is not suitable for separation of chlorogenic acid. Only NKA-9 and NKA-II resin are suitable. Comparatively speaking, NKA-9 resin has higher adsorption capacity and adsorption rate in static adsorption, and is easy to desorb, which has a good enrichment effect on chlorogenic acid.
2.4 Adsorption experiment of chlorogenic acid by resin
2.4. 1 dynamic adsorption curve Chlorogenic acid solution with a concentration of 3.0 mg ml-1was added to a glass chromatographic column filled with 200gNKA-9 macroporous resin. When the flow rate was 2.0 ml min-1(it can be expressed as BV/h, that is, the mobile phase volume flowing through the bed per hour was multiple of the resin bed volume. The results show that the resin can treat 7 ~ 8 times the volume of resin bed.
Fig./adsorption curve of chlorogenic acid by kloc-0/NKA-9 resin (omitted)
2.4.2 Influence of injection flow rate on adsorption In order to carry out effective adsorption, it is necessary to make the solid-liquid two phases have enough contact time in the process of resin adsorption. When the injection flow rate is too high, the resin can not adsorb chlorogenic acid, which leads to the decrease of adsorption rate. The injection flow rate is too small, although the adsorption is sufficient, the adsorption time is long and the efficiency is low. Chlorogenic acid solution with the concentration of 3.0 mg mL-1was passed through the column, and the relationship between the adsorption capacity of resin per unit time and different injection flow rates was investigated. The results show that the adsorption effect is the best when the flow rate is 2.0 ml min-1. See figure 2.
Fig. 2 Effect of injection flow rate on adsorption (omitted)
2.4.3 Effect of pH value of injection on adsorption Experiments were carried out at different pH values. The results showed that chlorogenic acid, as a polyhydroxy phenolic acid, was easily adsorbed under acidic conditions. Because chlorogenic acid exists in molecular form under acidic conditions, the adsorption of chlorogenic acid by resin is enhanced; Chlorogenic acid in lactone form is easy to hydrolyze under strong acid conditions; Under alkaline conditions, chlorogenic acid in ionic form is not easily adsorbed. As can be seen from Figure 3, when the pH value is 3, the adsorption effect is the best.
2.5 desorption experiment of chlorogenic acid by resin
2.5. 1 Selection of eluent After the compound is adsorbed by the resin, different eluents are selected according to the adsorption strength. Generally speaking, when adsorbents interact with adsorbents through intermolecular dispersion force, the adsorption force is weak and it is easy to elute. However, when adsorbents and adsorbents act through dipole force or hydrogen bond, the adsorption force is strong and it is not easy to elute. NKA-9 macroporous resin has strong polarity, and chlorogenic acid is a compound containing phenolic hydroxyl groups, which is easy to form hydrogen bonds with the resin. A more soluble solvent is needed to elute chlorogenic acid. In this experiment, ethanol aqueous solutions with different concentrations were selected as eluents, and the results are shown in Figure 4.
Fig. 3 Effect of pH value of injection on adsorption (omitted)
Fig. 4 Relationship between ethanol concentration of eluent and desorption rate (omitted)
In the experiment, ethanol solutions with different concentrations were used to elute, the content of chlorogenic acid in the eluent was determined, and the desorption rate was calculated. Among them, 50% and 70% ethanol solution can basically elute chlorogenic acid adsorbed by resin. The thin-layer analysis of the eluent shows that although the desorption rate of 70% ethanol solution is high, the impurity content in the eluent is also high, which can not achieve the separation effect of effective components, so 50% ethanol solution is selected as the eluent in the experiment.
2.5.2 Drawing elution curve After the sample clear liquid is adsorbed, it is eluted with 50% ethanol solution, and the elution flow rate is controlled at1.0bv/h, and the eluent is collected, and the content of chlorogenic acid in the eluent is determined, and the elution curve is drawn, as shown in Figure 5.
Fig. 5 elution curve of NKA-9 resin (omitted)
It can be seen that chlorogenic acid can be eluted by eluent with 3 times the volume of resin bed, and the elution peak is concentrated. Concentrating 50% ethanol eluent under reduced pressure, and drying at low temperature to obtain crude chlorogenic acid.
2.6. The crude chlorogenic acid was recrystallized with methanol and dried in vacuum to obtain white needle-like crystals. The structure of the product was confirmed by IR and 1HNMR analysis, and the content determined by HPLC was 97.95438 0%.
3 Conclusion
In this study, the extraction conditions of chlorogenic acid from Eucommia ulmoides leaves were optimized: Eucommia ulmoides leaves were extracted with deionized water at 80℃ for 65438±050min, and the sample solution was obtained after concentration, flocculation, impurity removal and adsorption decoloration. The sample solution was treated with NKA-9 macroporous resin to obtain crude chlorogenic acid, and the crude chlorogenic acid was recrystallized to obtain chlorogenic acid with a content of ≥97% and an extraction rate of ≥65%. Eucommia ulmoides Oliv China is rich in resources, and the technological conditions for extracting chlorogenic acid from Eucommia ulmoides Oliv. are optimized. Leaves provide basic data for the comprehensive utilization and industrial development of Eucommia ulmoides Oliv.
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(School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, Hubei 430073)