Highly selective separation and purification of chicoric acid from Echinacea purpurea by quality control methods in macroporous adsorption resin column chromatography

Chicoric acid is the main phenolic active ingredient in Echinacea purpurea (Asteraceae), best known for its immune‐enhancing ability, as well as used as a herbal medicine. To achieve further utilization of medicinal ingredients from E. purpurea, an efficient preparative separation of chicoric acid was developed based on macroporous adsorption resin chromatography. The separation characteristics of several different typical macroporous adsorption resins were evaluated by adsorption/desorption column experiments, and HPD100 was revealed as the optimal one, which exhibited that the adsorbents fitted well to the pseudo‐second‐order kinetics model and Langmuir isotherm model, and the optimal process parameters were obtained. The breakthrough curves could be predicted and end‐point could be determined early. Besides, the optimal elution conditions of chicoric acid can be achieved using the quality control methods. As a result, the purity of chicoric acid was increased 15.8‐fold (from 4 to 63%) after the treatment with HPD100. The process of the enrichment and separation of chicoric acid is considerate, because of its high efficiency and simple operation. The established separation and purification method of chicoric acid is expected to be valuable for further utilization of E. purpurea according to product application in pharmaceutical fields in the future.     

Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins

In present study, the performance and separation characteristics of five macroporous resins for the enrichment and purification of asiaticoside and madecassoside from Centella asiatica extracts have been evaluated. The adsorption and desorption properties of total triterpene saponins (80% purity) on macroporous resins including HPD100, HPD300, X-5, AB-8 and D101 have been compared. According to our results, HPD100 offered higher adsorption and desorption capacities and higher adsorption speed for asiaticoside and madecassoside than other resins. Column packed with HPD100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of asiaticoside and madecassoside from C. asiatica extracts. After the treatment with gradient elution on HPD100 resin, the content of madecassoside in the product increased from 3.9 to 39.7%, and the recovery yield was 70.4%; for asiaticoside the content increased from 2.0 to 21.5%, and the recovery yield was 72.0%. The results showed that HPD100 resin revealed a good ability to separate madecassoside and asiaticoside, and the method can be referenced for the separation of other triterpene saponins from herbal raw materials.     

Preparative separation and enrichment of four taxoids from Taxus chinensis needles extracts by macroporous resin column chromatography

An environment‐friendly method was established for the preparative separation and enrichment of four taxoids, namely 10‐deacetylbaccatin III (10‐DAB III), 7‐xylosyl‐10‐deacetyltaxol (7‐xyl‐10‐DAT), cephalomannine and paclitaxel from Taxus chinensis needles extracts. Characteristics of seven widely used macroporous resins for four taxoids were compared, AB‐8 resin offered better adsorption and desorption capacities than others. AB‐8 resin column chromatography was used to study the desorption process for four taxoids. The optimum parameters for desorption were 30% ethanol 5 RV for removing impurities, following 15 RV for 10‐DAB III, after the desorption of impurities with 35% ethanol 10 RV, 45% ethanol 30 RV for 7‐xyl‐10‐DAT, then 65% ethanol 10 RV for cephalomannine and paclitaxel, the flow rate was 6 RV/h. After separation on AB‐8 resin column chromatography, the contents of 10‐DAB III, 7‐xyl‐10‐DAT, cephalomannine and paclitaxel in the product reached 4.58, 13.17, 1.36 and 3.08%, respectively, which were 7.63‐, 3.68‐, 6.18‐ and 6.55‐fold to those in T. chinensis needles extracts. The recovery yields were 94.96, 77.32, 88.09 and 95.25%. In general, the AB‐8 resin column chromatography has the advantages of lower cost, high efficiency and simple procedure. Therefore, it may provide scientific references for the preparative separation and enrichment of taxoids from other T. species.     

Purification of spinosin from Ziziphi Spinosae Semen using macroporous resins followed by preparative high‐performance liquid chromatography

As a well‐known traditional Chinese medicine, Ziziphi Spinosae Semen has been used for treating anxiety and insomnia for a long time. Spinosin, the main active C‐glycoside flavonoid in Ziziphi Spinosae Semen, has attracted much attention because of its many pharmacological activities including strong hypnotic effects, anxiolytic‐like effects, and so on. In the present work, high‐purity spinosin was separated from Ziziphi Spinosae Semen using the HPD‐300 resin followed by preparative high‐performance liquid chromatography. The adsorption kinetics curve of spinosin on the HPD‐300 resin was studied and fitted well by the pseudo‐second‐order equation. The adsorption isotherms were also constructed and low temperature favored the adsorption reaction. The separation parameters were optimized using dynamic adsorption and desorption tests. After a one‐run treatment with HPD‐300 resin, the concentration of spinosin increased 11.8‐fold from 0.99 to 11.7% with a recovery yield of 80.4%. Furthermore, the purity of spinosin could surpass above 98% after separation by preparative high‐performance liquid chromatography and recrystallization with a recovery yield of 72.6%. The developed method was effective and suitable for the large‐scale preparation of spinosin. Moreover, it was confirmed that HPD‐300 resin could enable good selection for the enrichment of flavonoids from different plants.     

A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwa by combination of macroporous resin and counter‐current chromatography

In this paper, macroporous resin column chromatography and counter‐current chromatography (CCC) were applied for large‐scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning‐up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3‐hydroxyl‐genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3‐g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.     

Enrichment of total steroidal saponins from the extracts of Trillium tschonoskii Maxim by macroporous resin and the simultaneous determination of eight steroidal saponins in the final product by HPLC

An effective and simple method was established for the separation and enrichment of steroidal saponins from Trillium tschonoskii Maxim. The adsorption and desorption properties of seven macroporous resins were investigated. Among the tested resins, AB‐8 resin showed the best adsorption and desorption capacities. The adsorption of steroidal saponins on AB‐8 at 25°C was quite consistent with both the Freundlich isotherm model and the pseudo‐second‐order kinetics model. By optimizing the dynamic adsorption and desorption parameters, the content of steroidal saponins increased from 5.20% in the crude extracts to 51.93% in the final product, with a recovery yield of 86.67%. Furthermore, by scale‐up separation, the concentration and recovery of total steroidal saponins were 43.8 and 85.5%, respectively, which suggested that AB‐8 resin had great industrial and pharmaceutical potential because of its high efficiency and cost‐effectiveness. In addition, a high‐performance liquid chromatography method for the simultaneous determination of eight steroidal saponins was established for the first time, which was employed to qualitatively and quantitatively analyze the final product. Based on the methodological validation results, the high‐performance liquid chromatography method can be widely applied to the quality control of steroidal saponins from Trillium tschonoskii Maxim due to its excellent accuracy, stability, and repeatability.     

Enrichment and purification of deoxyschizandrin and γ-schizandrin from the extract of Schisandra chinensis fruit by macroporous resins

In present study, the performance and separation characteristics of 21 macroporous resins for the enrichment and purification of deoxyschizandrin and γ-schizandrin, the two major lignans from Schisandra chinensis extracts, were evaluated. According to our results, HPD5000, which adsorbs by the molecular tiers model, was the best macroporous resin, offering higher adsorption and desorption capacities and higher adsorption speed for deoxyschizandrin and γ-schizandrin than other resins. Columns packed with HPD5000 resin were used to perform dynamic adsorption and desorption tests to optimize the technical parameters of the separation process. The results showed that the best adsorption time is 4 h, the rate of adsorption is 0.85 mL/min (4 BV/h) and the rate of desorption is 0.43 mL/min (2 BV/h). After elution with 90% ethanol, the purity of deoxy-schizandrin increased 12.62-fold from 0.37% to 4.67%, the purity of γ-schizandrin increased 15.8-fold from 0.65% to 10.27%, and the recovery rate was more than 80%.     

Separation and purification of hydroxytysol and oleuropein from Olea europaea L. (olive) leaves using macroporous resins and a novel solvent system

The separation and purification of hydroxytysol and oleuropein from Olea europaea L. (olive) using a macroporous resin with a novel solvent system was systematically investigated. Static adsorption experiments with BMKX–4 resin revealed that the experimental data of both hydroxytysol and oleuropein fitted best to the pseudo‐second‐order kinetic and Freundlich isotherm models. The thermodynamic parameters indicated spontaneous and exothermic adsorption processes. The novel solvent system, composed of n–hexane:ethyl acetate:methanol:water in a (v/v/v/v) ratio of 1:9:1:9, had two phases (upper and lower). The separation and purification parameters of hydroxytysol and oleuropein were optimized using dynamic adsorption/desorption on a column packed with BMKX–4 resin. The effects of flow rates and volumes of the upper and lower phases on the separation efficiency were systematically studied. Under optimal conditions, the fraction of hydroxytysol in the final product increased by 6.34‐fold from 0.46 to 2.96%, with a yield rate of 88.58% w/w, while that of oleuropein increased 4.17‐fold from 11.40 to 47.59%, with a 93.31% w/w yield rate. These results may be help in selecting a suitable eluent for improved separation of macroporous adsorption resins.     

大孔吸附树脂富集板蓝根中(R,S)-告依春工艺研究

采用静态吸附法考察了D101、AB-8、NKA-2、NKA-9、HPD 100、HPD600等6种大孔吸附树脂对(R,S)-告依春的吸附及解吸性能,筛选出效果最佳的AB-8树脂,并对其进行动态考察.最佳富集条件为:上样液pH 6,生药质量-体积浓度为0.200g/mL,解吸液为2BV量70％乙醇,在优化条件下(R,S)-告依春在浸膏中含量可从0.76％提高到12.48％.结果表明,AB-8型大孔吸附树脂可用来从板蓝根水提取液中富集(R,S)-告依春.     

Development of a process for separation of mogroside V from Siraitia grosvenorii by macroporous resins

A separation method was developed for the preparative separation and enrichment of the non-caloric sweetener mogroside V from Siraitia grosvenorii. The adsorption properties of six macroporous resins were evaluated. Results showed that HZ 806 resin offered the best adsorption and desorption capacities. Based on the adsorption experiments on HZ 806, the adsorption data were found to fit the Freundlich model well. The pseudo-second-order kinetic model showed the highest correlation with the experimental results. Separation was performed with deionized water and 40% aqueous ethanol solution as mobile phases. In a typical run, 100 g of herb was processed and 3.38 g of mogroside V with a purity of 10.7% was harvested. This separation method provided a 15.1-fold increase in the purification factor from 0.5% to 10.7%. The present study showed that HZ 806 resins were effective for the separation and enrichment of mogroside V from S. grosvenorii.     

Separation of salvianic acid A from the fermentation broth of engineered Escherichia coli using macroporous resins

Salvianic acid A (also known as danshensu) is a plant‐derived polyphenolic acid, and has a variety of physiological and pharmacological activities. Our laboratory previously constructed an unprecedented artificial biosynthetic pathway in Escherichia coli and established the fermentation process to produce salvianic acid A. Here, we developed an efficient method for separating salvianic acid A from the fermentation broth of engineered Escherichia coli by macroporous resins. Among ten tested macroporous resins, the static and dynamic adsorption/desorption experiments demonstrated that X5 resin was the best to separate salvianic acid A from fermentation broth. Other parameters during static and dynamic procedures were also investigated. Under the optimum separation conditions, the average adsorption capacity of SAA were 10.66±0.54 mg/g dry resin and the desorption ratio was 85.6±4.1%. The purity and recovery yield of salvianic acid A in the final dry product were 90.2±1.5 and 81.5±2.3%, respectively. The results show that adsorption separation with macroporous resin X5 was an efficient method to prepare salvianic acid A from fermentation broth. This work will benefit the development and application of plant‐derived salvianic acid A and its derivatives.     

Separation of injectable salidroside by column chromatography of macroporous resins for treating myocardial ischemia

The objective of the present study is to develop a method for large-scale separating and purifying salidroside from rhodiola kirilowii roots and for preparing injectable medicinal ingredient.Crude extract of salidroside was prepared by water-ethanol system,and purified by column chromatography of macroporous resins.Static adsorption and desorption studies were performed on six kinds of macroporous resins,and SP825 resin was chosen,followed by optimizing process parameters.The optimum sample volume,feed concentration,ratio of diameter to height,and feeding flow rate were 1.5 bed volumes(BV),15 mg/mL,1:10 and 1 BV/h,respectively.Dynamic desorption was performed consecutively with 8 BV of distilled water,3 BV of 5% ethanol and 8 BV of 10% ethanol at a flow rate of 2 BV/h.After three cycles in separating 3.5 tons of rhodiola kirilowii roots,salidroside purity was increased from 3.4% in the crude extract to 93.6% in purified salidroside product.This study provides a novel method to separate salidroside for injectable use.     

Preparative enrichment and separation of astragalosides from Radix Astragali extracts using macroporous resins

The enrichment and separation of astragalosides I–IV (AGs I–IV) were studied on eight macroporous resins in the present study. SA‐3 resin offered the best adsorption and desorption capacities for AGs I–IV than other resins. The models of adsorption kinetics were investigated in order to elucidate the mechanism of adsorption. The pseudo‐second‐order model was the better choice than the pseudo‐first‐order model to describe the adsorption behavior of AGs I–IV onto SA‐3 resin. The equilibrium experimental data were well fitted to Langmuir and Freundlich isotherms. SA‐3 resin adsorption chromatography tests were carried out to optimize the separation process of AGs I–IV from Radix Astragali extracts. With the optimum parameters for adsorption and desorption, the contents of AGs I–IV were 8.78‐, 11.60‐, 10.52‐ and 11.28‐fold increased with the recovery yields being 65.88, 90.92, 84.25 and 94.17%, respectively. The preparative enrichment and separation of AGs I–IV from Radix Astragali extracts can be easily and effectively achieved by SA‐3 resin adsorption chromatography. The developed methodology can also be referenced for the separation of other active constituents from herbal materials and manufacture of Radix Astragali products.     

Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis A. Bor. The crude salidroside was obtained by extraction with methanol from Rhodiola sachalinensis A. Bor. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was successfully performed yielding salidroside (32 mg) at 98% purity from 250 mg of the crude extract in a one-step separation.     

大孔吸附树脂分离纯化异甘草素的研究

研究大孔吸附树脂分离纯化异甘草素的工艺条件及参数。通过研究HPD-600、D4020、D101、AB-8、NKA-II、AL-2和NKA-9树脂对异甘草素的吸附和解吸附能力,筛选最佳树脂为AB-8,并研究了其对异甘草素的吸附和解吸附性能,确定了最佳的吸附与解吸附工艺参数,吸附:pH=5,室温,流速1.5BV/h,溶液处理量为5BV;脱附:洗脱剂为70%的乙醇溶液,流速1BV/h,洗脱剂用量4.5BV。异甘草素样品溶液经AB-8树脂吸附与脱附后回收率为76.7%,纯度由2.02%提高到29.1%,提高了14.4倍。实验结果表明,AB-8树脂对异甘草素的吸附量大,脱附容易,可以应用于异甘草素的分离纯化。     

A green and efficient protocol for large‐scale production of glycyrrhizic acid from licorice roots by combination of polyamide and macroporous resin adsorbent chromatography

A green and efficient method for large‐scale preparation of glycyrrhizic acid from licorice roots was developed by combination of polyamide and macroporous resin. The entire preparation procedure consisted of two simple separation steps. The first step is to use polyamide resin to remove licorice flavoniods from the licorice crude extract. Subsequently, various macroporous resins were tried to purify glycyrrhizic acid, and HPD‐400 showed the most suitable adsorption and desorption properties. Under the optimized conditions, a large‐scale preparation of glycyrrhizic acid from licorice roots was carried out. A 20 kg raw material produced 0.43 kg of glycyrrhizic acid using green aqueous ethanol as the solvent. The purity of glycyrrhizic acid was increased from 11.40 to 88.95% with a recovery of 76.53%. The proposed method may be also extended to produce large‐scale other triterpenoid saponins from herbal materials.     

Separation and purification of flavonoid from Taxus remainder extracts free of taxoids using polystyrene and polyamide resin

An efficient separation process of flavonoid from Taxus wallichiana var. mairei remainder extracts free of taxoids was developed in this study. AB‐8 macroporous resin and polyamide resin offered the fine adsorption capacity, and its adsorption rate at 30°C fitted well to the Langmuir and Freundich isotherms. Resin dynamic adsorption and desorption experiments were conducted to optimize the separation process of total flavonoids from T. wallichiana var. mairei remainder extracts free of taxoids. The optimum parameters for adsorption by AB‐8 resin were as follows: (1) the concentration of flavonoids in a sample solution of 5.61 mg/mL with a processing volume of 2 bed volume (BV) (60 mL); (2) for desorption, ethanol–water (80:20, v/v), with 6 BV as an eluent at a flow rate of 2 BV/h. After a one‐run treatment with AB‐8 resin, the content of flavonoids was increased 5.10‐fold from 4.05 to 20.65%. The optimum parameters for adsorption by polyamide resin were as follows: processing volume of 2 BV (30 mL); for desorption, ethanol–water (70:30, v/v), with 8 BV as an eluent at a flow rate of 2 BV/h. After one‐run treatment with polyamide resin, the content of total flavonoids increased from 20.65 to 65.21%. The method will provide a potential approach for large‐scale separation and purification of flavonoid for its wide pharmaceutical use.     

Preparative separation of vitexin and isovitexin from pigeonpea extracts with macroporous resins

Vitexin and isovitexin are a pair of isomeric compounds known as the major constituents in pigeonpea leaves and possess various pharmacological activities. In the present study, the preparative separation of vitexin and isovitexin with macroporous resins (Nankai Hecheng S & T, Tianjin, China) was studied. The performance and adsorption characteristics of eight macroporous resins including ADS-5, ADS-7, ADS-8, ADS-11, ADS-17, ADS-21, ADS-31 and ADS-F8 have been evaluated. The research results indicate that ADS-5 resin is most appropriate for the separation of vitexin and isovitexin. Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures, and the equilibrium experimental data were well fitted to the two isotherms. Column packed with ADS-5 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. The optimum parameters for adsorption were as follows: the concentration of vitexin and isovitexin in sample solution: 0.22 and 0.40mg/mL, respectively, processing volume: 3 BV, flow rate: 1mL/min, pH 4, temperature: 25 degrees C; for desorption: ethanol-water (40:60, v/v), 5 BV as an eluent, flow rate: 1mL/min. After one run treatment with ADS-5 resin, the contents of vitexin and isovitexin were increased 4.07-fold and 11.52-fold from 0.86%, 1.53% to 3.50% and 17.63%, the recovery yields were 65.03% and 73.99%, respectively. In conclusion, the preparative separation of vitexin and isovitexin can be easily and effectively achieved via adsorption and desorption on ADS-5 resin, and the method can be referenced for the separation of other flavone C-glucosides from herbal materials.     

大孔吸附树脂对酯型儿茶素吸附性能的研究

系统研究了AB-8,PA,HPD600,NKA-9,NKA-II等5种大孔吸附树脂对EGCG的吸附性能。结果表明,这5种大孔吸附树脂对EGCG的吸附效率随AB-8,PA,HPD600,NKA-9,NKA-II的顺序依次减小。选择大孔吸附树脂PA为吸附剂,用PA对EGCG、ECG、GCG进行静态和动态吸附实验以及解吸剂的选择实验,研究了吸附速率曲线,确定了最佳吸附流速,根据解吸效果和绿色提取的需要,决定选用无毒有机溶剂C作为解脱剂。     

Authentication of Rhodiola rosea,Rhodiola quadrifida and Rhodiola rosea liquid extract from the Ukrainian market using HPTLC chromatographic profiles

Abstract

Rhodiola rosea and Rhodiola quadrifida are widely distributed and sold in Eastern Europe. The purpose of this paper was to identify R.rosea, R.quadrifida and Rhodiola rosea liquid extract (RRLE) in the Ukrainian market and bring out adulteration cases using chromatographic characterisation by HPTLC. The multiple samples of R.rosea, R.quadrifida and RRLE were compared; the optimal chromatographic conditions for identification of R.rosea and RRLE based on the presence of rosavins and salidroside as well as for identification of R.quadrifida based on the presence of salidroside were proposed; the specific HPTLC fingerprints were obtained; the acceptance criteria for each product were set. The adulteration cases for R.rosea and RRLE samples were established. The dependence on handling R.rosea and presence of rosavins was determined. It was assumed that low-quality raw materials or inefficient technology process were used for RRLE. The consistency of HPTLC fingerprints for R.quadrifida samples was established.