Analyzing chemical composition of Sargentodoxae caulis water extract and their hypouricemia effect in hyperuricemic mice.

Hyperuricemia (HUA) is a metabolic disease characterized by the increase of serum uric acid (UA) level. Sargentodoxae Caulis (SC) is a commonly used herbal medicine for the treatment of gouty arthritis, traumatic swelling, and rheumatic arthritis in clinic. In this study, a total of fifteen compounds were identified in SC water extract using UHPLC-Q-TOF-MS/MS, including three phenolic acids, seven phenolic glycosides, four organic acids, and one lignan. Then, to study the hypouricemia effect of SC, a HUA mouse model was induced using a combination of PO, HX, and 20% yeast feed. After 14 days of treatment with the SC water extract, the levels of serum UA, creatinine (CRE), blood urea nitrogen (BUN) were reduced significantly, and the organ indexes were restored, the xanthine oxidase (XOD) activity were inhibited as well. Meanwhile, SC water extract could ameliorate the pathological status of kidneys and intestine of HUA mice. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting results showed that SC water extract could increase the expression of ATP binding cassette subfamily G member 2 (ABCG2), organic cation transporter 1 (OCT1), organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3), whereas decrease the expression of glucose transporter 9 (GLUT9). This study provided a data support for the clinical application of SC in the treatment of HUA.

Screening and characterization of potential anti-gout components from Polygonum cuspidatum by integration off-line two-dimensional liquid chromatography-mass spectrometry with affinity ultrafiltration and on-line HPLC-ABTS.

Polygonum cuspidatum (P. cuspidatum) is a traditional herbal medicine with a long history and proven efficacy in treating gout. However, due to the complexity of composition and extensive content distribution, the substance basis of its anti-gout effectiveness is still unclear. A strategy was proposed via integrating off-line two-dimensional liquid chromatography (2D-LC) and targeted rapid screening technology based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC/MS) and on-line high-performance liquid chromatography-2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (HPLC-ABTS) to accomplish high coverage and high throughput screening of anti-gout components from P. cuspidatum. As a result, twenty components were screened from P. cuspidatum extract with both xanthine oxidase (XOD) inhibitory activity and free radical scavenging activity, then were preliminarily identified by high-resolution electrospray ionization-quadrupole-time-of-flight mass spectrometer (ESI-Q-TOF/MS). The screened results were verified by the in vitro assays. Meanwhile, molecular docking further elucidated that the screened bioactive ingredients had favourable binding capabilities with XOD. The performance of this study can achieve high efficiency and high coverage screening of the anti-gout components from P. cuspidatum, which provides methodology and strategy support for the rapid screening of bioactive ingredients from complex medicinal plants.

Structure and properties of acidic polysaccharides isolated from Massa Medicata Fermentata: Neuroprotective and antioxidant activity.

Massa Medicata Fermentata (MMF) is a fermented food with therapeutic effects. Previous studies suggested that after stir-frying, the uronic acid content in MMF crude polysaccharides increases, and the pH value decreases, which is caused by the change in acidic polysaccharides. However, the detailed physicochemical properties and structure-activity correlation of the acidic polysaccharides in MMF have not been fully explored. In this study, two acidic polysaccharides (SMMFAP and CMMFAP) were isolated from the MMF and its stir-fried product, respectively. Their structural characteristics and bioactivities were comparatively studied, and the structure-activity correlation was examined. Our findings revealed that the SMMFAP had a higher average Mw and higher Gal and Man content than the CMMFAP. Both the SMMFAP and CMMFAP were mainly composed of Xyl, Man, and Gal residues, whereas the CMMFAP had fewer linkage types. Additionally, the CMMFAP exhibited stronger neuroprotective activity than the SMMFAP owing to its higher content of 1,6-linked-Galp, while the SMMFAP exhibited better antioxidant activity, which might be related to its higher average Mw. Our findings suggest that acidic polysaccharides may be the active substances that cause differences in effectiveness between the sheng and chao MMF. Furthermore, the research qualified the SMMFAP and CMMFAP with different potential applications.

Isolation, characterization, trypsin inhibition, liver protective and antioxidant activities of arabinoxylan from Massa Medicata Fermentata and its processed products.

Massa Medicata Fermentata (MMF) is a traditional Chinese medicine widely used in feed additives and human medicine. In this study, two neutral polysaccharides (SMMFP-1 and CMMFP-1) were isolated from two forms of MMF (sheng and chao MMF), and their structural characteristics and bioactivities were studied. The results showed that CMMFP-1 had higher average Mw compared with that of SMMFP-1. SMMFP-1 had a lower proportion of Ara, Xyl, GalA, and GlcA, but higher levels of Fuc, Gal, Man, and GulA. Compared with CMMFP-1, SMMFP-1 had a triple helix structure. SMMFP-1 had a layered structure, whereas CMMFP-1 had a curly layered structure. More glycosidic linkage types were found in SMMFP-1 than in CMMFP-1, and SMMFP-1 had a greater number of side chains. More importantly, SMMFP-1 showed better trypsin inhibition activity in vitro, liver-protective activity in vivo, and stronger antioxidant activity in vivo than CMMFP-1. Thus, arabinoxylans may be one of the active substances for different efficacies between MMF and its processed product. The results of this study facilitate the exploration of the correlation between the structural characteristics and biological functionalities of MMF arabinoxylans. Moreover, a theoretical basis is established for further study of the unique properties of arabinoxylans and their applications.

Integration of metabolomics and network pharmacology to reveal the protective mechanism underlying Wogonoside in acute myocardial ischemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional medicine, both Scutellaria baicalensis Georgi (SBG) and the traditional formulas composed of it have been used to treat a wide range of diseases, including cancer and cardiovascular. Wogonoside (Wog) is the biologically active flavonoid compound extracted from the root of SBG, with potential cardiovascular protective effects. However, the mechanisms underlying the protective effect of Wog on acute myocardial ischemia (AMI) have not yet been clearly elucidated. AIM OF THE STUDY: To explore the protective mechanism of Wog on AMI rats by comprehensively integrating traditional pharmacodynamics, metabolomics, and network pharmacology. METHODS: The rat was pretreatment with Wog at a dose of 20 mg/kg/d and 40 mg/kg/d once daily for 10 days and then ligated the left anterior descending coronary artery of rats to establish the AMI rat model. Electrocardiogram (ECG), cardiac enzyme levels, heart weight index (HWI), Triphenyltetrazolium chloride (TTC) staining, and histopathological analyses were adopted to evaluate the protective effect of Wog on AMI rats. Moreover, a serum metabolomic-based UHPLC-Q-Orbitrap MS approach was performed to find metabolic biomarkers and metabolic pathways, and network pharmacology analysis was applied to predict targets and pathways of Wog in treating AMI. Then, the network pharmacology and metabolomic results were integrated to elucidate the mechanism of Wog in treating AMI. Finally, RT- PCR was used to detect the mRNA expression levels of PTGS1, PTGS2, ALOX5, and ALOX15 to validate the result of integrated metabolomics and network analysis. RESULTS: Pharmacodynamic studies suggest that Wog could effectively prevent the ST-segment of electrocardiogram elevation, reduce the myocardial infarct size, heart weight index, and cardiac enzyme levels, and alleviate cardiac histological damage in AMI rats. Metabolomics analysis showed that the disturbances of metabolic profile in AMI rats were partly corrected by Wog and the cardio-protection effects on AMI rats involved 32 differential metabolic biomarkers and 4 metabolic pathways. In addition, the integrated analysis of network pharmacology and metabolomics showed that 7 metabolic biomarkers, 6 targets, and 6 crucial pathways were the main mechanism for the therapeutic application of Wog for AMI. Moreover, the results of RT-PCR showed that PTGS1, PTGS2, ALOX5, and ALOX15 mRNA expression levels were reduced after treatment with Wog. CONCLUSION: Wog exerts cardio-protection effects on AMI rats via the regulation of multiple metabolic biomarkers, multiple targets, and multiple pathways, our current study will provide strong scientific evidence supporting the therapeutic application of Wog for AMI.

An efficient Pt@MXene platform for the analysis of small-molecule natural products.

Small-molecule (m/z<500) natural products have rich biological activity and significant application value thus need to be effectively detected. Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has become a powerful detection tool for small-molecule analysis. However, more efficient substrates need to be developed to improve the efficiency of SALDI MS. Thus, platinum nanoparticle-decorated Ti3C2 MXene (Pt@MXene) was synthesized in this study as an ideal substrate for SALDI MS in positive ion mode and exhibited excellent performance for the high-throughput detection of small molecules. Compared with using MXene, GO, and CHCA matrix, a stronger signal peak intensity and wider molecular coverage was obtained using Pt@MXene in the detection of small-molecule natural products, with a lower background, excellent salt and protein tolerance, good repeatability, and high detection sensitivity. The Pt@MXene substrate was also successfully used to quantify target molecules in medicinal plants. The proposed method has potentially wide application.

[Application of bioassay in quality evaluation of Chinese medicine:a review].

The quality of Chinese medicine is the foundation of the clinical effects and industrial development. Component analysis ensures the consistency and stability of medicinals, but fails to evaluate the clinical efficacy. Bioassay is an analytical method to evaluate the effect of a substance on living organisms, tissues, or cells, which is an optimal option for assessing the quality of Chinese medicine. Bioassay of Chinese medicine starts early but progresses slowly. At the moment, it has attracted the interest of scholars. However, no systematic research is available. This study aims to summarize the research on the application of bioassay in quality evaluation of Chinese medicine, focusing on the application of key techniques and experimental systems in bioassay in heat-clearing and blood-activating and stasis-eliminating Chinese medicine and the common problems. Meanwhile, suggestions were proposed in terms of the association with clinical efficacy and chemical analysis and the status quo of biological assay. This study is expected to promote the study and application of bioassay.

MeJA regulates the accumulation of baicalein and other 4'-hydroxyflavones during the hollowed root development in Scutellaria baicalensis.

The dried roots of Scutellaria baicalensis are important traditional Chinese medicine used to treat liver and lung inflammation. An anomalous structure, hollowed root, was discovered in perennial cultivated Scutellaria baicalensis. The presence of the hollow may change the contents of bioactive metabolites, such as baicalein, and other 4'-hydroxyflavones in Scutellaria baicalensis roots, but the relationship between the hollowed root and bioactive metabolite contents is poorly understood. In this study, we identified the anatomical structure of the hollowed root and detected differentially accumulating flavonoid metabolites and enzymes related to 4'-hydroxyflavone biosynthesis in 3-year-old roots with a hollow. We confirmed that methyl jasmonate (MeJA) induced the accumulation of 4'-hydroxyflavones and the expression of enzymes related to 4'-hydroxyflavone biosynthesis in hydroponically cultured Scutellaria baicalensis roots. The development of the hollowed root were divided into 4 stages. The 4'-hydroxyflavone contents and expression of enzymes related to 4'-hydroxyflavone biosynthesis increased synchronously with the content of MeJA during the development of hollowed root. Pathogen and programed-cell-death related genes were induced during hollowed root development. Taken together, our results provide novel insight into the importance of MeJA in the development of hollowed root and the accumulation of 4'-hydroxyflavones in Scutellaria baicalensis roots. Our results suggest that a pathogen and senescence are the two major causes for the development of hollowed root in Scutellaria baicalensis roots.

Extraction of Flavonoids from Scutellariae Radix using Ultrasound-Assisted Deep Eutectic Solvents and Evaluation of Their Anti-Inflammatory Activities.

Deep eutectic solvents (DESs) play important roles in the extraction of active constituents in traditional Chinese medicine. Ultrasound-assisted DES has been used to extract flavonoids from Scutellaria baicalensis. Using the contents of scutellarin, baicalin, baicalein, wogonoside, wogonin, and oroxylin A as quantitative indices, different kinds of DESs have been optimized for extraction and betaine/acetic acid has shown the highest yield. The Box-Behnken response surface method (RSM) was utilized to select the extraction conditions with the highest yields. The optimal extraction conditions were as follows: the molar ratio of betaine/acetic acid was 1:4, the water content was 40%, the solid/liquid ratio was 1:100 g/mL, the extraction temperature was 52 °C, and the extraction time was 23 min. Compared with traditional reflux extraction using 70% ethanol as the solvent, ultrasound-assisted DES has a shorter extraction time and higher yields. Furthermore, anti-inflammatory activities of the two extracts by ultrasound-assisted DES and reflux were compared using RAW264.7 cells and the methyl thiazolyl tetrazolium (MTT) method, and they showed equal anti-inflammatory activities. The results demonstrated that the ultrasound-assisted DES method for extraction of flavonoids from scutellariae radix is simple, green, efficient, and reproducible. This research provides good method guides for the rapid and efficient extraction of flavonoids from natural sources.

A spatially-resolved approach to visualize the distribution and biosynthesis of flavones in Scutellaria baicalensis Georgi.

Imaging the spatial distributions and dynamics of flavones in heterogeneous plant tissues is significant for our understanding of plant metabolism. Here, we proposed a spatially-resolved approach to map the locations and biosynthesis of flavones in S. baicalensis. A total of 11 flavones, 5 flavone glycosides, 6 carbohydrates, and a variety of flavone synthesis-related metabolites were imaged. Most of these flavone-related metabolites presented stronger ion intensities in root phloem. The biosynthetic network of flavones and their glycosides in S. baicalensis were visualized for the first time. Moreover, we characterized the region-specific activities of four crucial enzymes in flavone synthesis pathway, including l-phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate coenzyme A ligase, and flavone synthase. In line with the spatial characteristic of flavones, all these four enzymes exhibit higher activity in the root phloem of S. baicalensis. The combination of spatially-resolved metabolites and enzymes information greatly broadens our understanding of flavone biosynthetic network.

Isolation and purification of six aristolochic acids with similar structures from Aristolochia manshuriensis Kom stems by pH-zone-refining counter-current chromatography.

Aristolochic acids (AAs), the major components in Aristolochia manshuriensis Kom stems (AMK), may cause Chinese herb nephropathy during clinical application. Therefore, it is necessary to distinguish AMK from other herbs and Chinese medicines using AAs with high purity as standards. So, an efficient method for separation and purification of AAs is required because of their similar structures. In this study, six AAs with purities of >98% were obtained by pH-zone-refining counter-current chromatography (PZRCCC) in a single run. The optimum two-phase solvent system was petroleum ether-ethyl acetate-methanol-water (3:7:3:7, v/v). Triethylamine (10 mM) was added to the aqueous mobile phase and trifluoroacetic acid (10 mM) to the organic stationary phase. As a result, 9.7 mg aristolochic acid IIIa, 12.0 mg aristolochic acid IVa, 32.2 mg aristolochic acid II, 103.7 mg aristolochic acid I, 24.6 mg aristolic acid II, and 26.1 mg aristolic acid I were obtained from 800 mg AAs crude extract. The elution order of AAs during PZRCCC separation corresponded with the pKa values and hydrophobicities of the target compounds. PZRCCC is an efficient method for isolation of AAs with similar structures.

Purification of pyrethrins from flowers of Chrysanthemum cineraraeflium by high-speed counter-current chromatography based on coordination reaction with silver nitrate.

Pyrethrum extract is a natural insecticide that has been used worldwide for centuries, with pyrethrins being considered active ingredients. Their purification is difficult due to their chemical structural similarity and instability to light. In the present study, a coordination complex high-speed counter-current chromatography was used to separate pyrethrins from pyrethrum extract. The two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (10:2:10:2, v/v/v/v) with 0.30 mol/L silver nitrate was selected for separation. As a result, five pyrethrins including cinerin II (31.2 mg), pyrethrin II (43.9 mg), jasmalin II (39.1 mg), pyrethrin I (32.4 mg), and jasmalin I (16.0 mg) were obtained from 400 mg crude extract in 6 h by one-step HSCCC separation, with purities over 95%. The isolated compounds were identified by MS, 1H-NMR and 13C-NMR spectroscopy. The results demonstrated that the addition of silver nitrate in the two-phase solvent system of HSCCC significantly increased the HSCCC separation factor of pyrethrins. HSCCC is a rapid method for purification of pyrenthrins from pyrethrum extract. The established method may also be applied to separate analogous compounds from natural products in which the chemical structure differs in the position or numbers of alkenes.

Evaluation of a Nondestructive NMR and MRI Method for Monitoring the Drying Process of Gastrodia elata Blume.

Gastrodia elata Blume (G. elata) is a prominent traditional herb and its dry tuber is officially listed in the Chinese Pharmacopoeia. To ensure the quality of dried G. elata, the establishment of a nondestructive and convenient method to monitor the drying process is necessary. In this study, a nondestructive low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) method was introduced to monitor the drying process of G. elata. Three water states (bound, immobilized, and free) in G. elata samples were investigated through multiexponential fitting and inversion of the NMR data. The variation and distribution of the three water states during drying were monitored by LF-NMR, and the spatial distribution of water and internal structural changes were analyzed by MRI. Linear analysis of the moisture content, L* (lightness), b* (yellowness), and NMR parameters showed good correlations among them. Furthermore, partial least squares regression (PLSR) model analysis, which takes into account all NMR parameters, also showed good correlations among these parameters. All results showed that LF-NMR was feasible and convenient for monitoring moisture content. Therefore, LF-NMR and MRI could be used to monitor the moisture content nondestructively in the drying process of Chinese traditional herbs.

Ultra-high-pressure-assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high-speed counter-current chromatography.

Ultra-high-pressure extraction combined with high-speed counter-current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra-high-pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid-liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra-high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (3:7:5:5, v/v) was used for high-speed counter-current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one-step separation. This research demonstrated that ultra-high-pressure extraction combined with high-speed counter-current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.

Four new phenolic constituents from the rhizomes of Gastrodia elata Blume.

Four new gastrodin derivatives containing a trans-cinnamoyl unit (1-4) and nine known compounds (5-13) were isolated from the rhizomes of Gastrodia elata Blume. All these compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine-induced cell death, and compounds 7 and 12 showed potent activities with EC50 values of 10.5 and 10.2 µM, respectively.

Effect of inorganic salt on partition of high-polarity parishins in two-phase solvent systems and separation by high-speed counter-current chromatography from Gastrodia elata Blume.

Parishins are high-polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two-phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two-phase solvent system composed of butyl alcohol/acetonitrile/near-saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high-speed counter-current chromatography. Fractions obtained from high-speed counter-current chromatography were subjected to semi-preparative high-performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two-phase solvent system in high-speed counter-current chromatography was a suitable approach for the purification of high-polarity compounds.

Chemical Constituents from Scindapsus officinalis (Roxb.) Schott. and Their Anti⁻Inflammatory Activities.

One new monoterpene glycoside (1), one new phenyl glycoside (2), one new caffeoyl derivative (3), were isolated from Scindapsus officinalis (Roxb.) Schott., along with four known compounds (4⁻7). Structures of the isolated compounds were elucidated by extensive analysis of spectroscopic data, especially 2D NMR data and comparison with literatures. All isolates were evaluated for anti-inflammatory activity against nitric oxide (NO) production in vitro. Compounds 3 and 7 exhibited moderate inhibitory effects on NO production with IC50 values of 12.2 ± 0.8 and 18.9 ± 0.3 µM, respectively.

Preparative separation of caffeoylquinic acid isomers from Lonicerae japonicae Flos by pH-zone-refining counter-current chromatography and a strategy for selection of solvent systems with high sample loading capacities.

Caffeoylquinic acid derivatives exhibit anti-inflammatory, antioxidant, and antibacterial activities. We successfully applied pH-zone-refining counter-current chromatography (pH-ZRCCC) to separation of isomeric caffeoylquinic acids from Lonicerae japonicae Flos using a two-phase solvent system composed of ethyl acetate-n-butanol-acetonitrile-water (3:1:1:5, v/v/v/v). Trifluoroacetic acid (10 mM) was added to the upper phase as a retainer and ammonium hydroxide (10 mM) was added to the lower phase as an eluter. As a result, 167.8 mg of chlorogenic acid, 15.9 mg of isochlorogenic acid B, 103.4 mg of isochlorogenic acid C, and 156.0 mg of isochlorogenic acid A were obtained from 1.2 g of crude extract, and all compound purities were over 96%. In addition, by comparing the numeric values of partition coefficient of compounds, it was found that larger differences between the K values of adjacent compounds in the solvent systems resulted in higher sample loading capacities. pH-ZRCCC method is an efficient preparative separation of isomeric caffeoylquinic acids from natural products.

Separation of Seven Polyphenols from the Rhizome of Smilax glabra by Offline Two Dimension Recycling HSCCC with Extrusion Mode.

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. Then, HSCCC was applied to separate polyphenols from the two fractions using a solvent system consisting of petroleum ether-ethyl acetate-methanol-water (1:3:0.5:5, v/v). Fifty milligrams of Fr.1 was separated by conventional HSCCC, yielding 5-O-caffeoylshikimic acid (1, 15.8 mg) and taxifolin (2, 4.8 mg). Offline 2D R-HSCCC with extrusion mode was used to separate Fr.2, and astilbin (4, 37.3 mg), neoisoastilbin (5, 8.8 mg), engeletin (7, 7.9 mg), and a mixture of two polyphenols were obtained from 100 mg of Fr.2. The mixture of two polyphenols was further separated by pre-HPLC, yielding neoastilbin (3, 15.2 mg) and isoastilbin (6, 9.9 mg). The purities of these seven compounds were all over 96.0%. Their structures were identified by MS and NMR. The results demonstrated that the strategy based on offline 2D R-HSCCC with extrusion mode was a powerful tool to separate the main compounds from the rhizome of Smilax glabra and valued for the preparative separation compounds with broad K-values and similar structures.

Semi-Preparative Separation of 10 Caffeoylquinic Acid Derivatives Using High Speed Counter-Current Chromatogaphy Combined with Semi-Preparative HPLC from the Roots of Burdock (Arctium lappa L.).

Burdock roots are healthy dietary supplements and a kind of famous traditional Chinese medicine, which contains large amounts of caffeoylquinic acid derivatives. However, little research has been reported on the preparative separation of these compounds from burdock roots. In the present study, a combinative method of HSCCC and semi-preparative HPLC was developed for the semi-preparative separation of caffeoylquinic acid derivatives from the burdock roots. The ethyl acetate extract of burdock roots was first fractionated by MCI macroporous resin chromatography and give three fractions (Fr. 1-3) from the elution of 40% methanol. Then, these three fractions (120 mg) were separately subjected to HSCCC for purification with the solvent system composed of petroleum ether-ethyl acetate-methanol-water at different volume ratios, and the mixtures were further purified by semi-preparative HPLC. As a result, a total of eight known caffeoylquinic acid derivatives including 3-O-caffeoylquinic acid (32.7 mg, 95.7%), 1,5-O- dicaffeoylquinic acid (4.3 mg, 97.2%), 3-O-caffeoylquinic acid methyl ester (12.1 mg, 93.2%), 1,3-O-dicaffeoylquinic acid (42.9 mg, 91.1%), 1,5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (4.3 mg, 84.5%), 4,5-O-dicaffeoylquinic acid (5.3 mg, 95.5%), 1,5-O-dicaffeoyl-3-O-succinylquinic acid (8.7 mg, 93.4%), and 1,5-O-dicaffeoyl-4-O-succinylquinic acid (1.7 mg, 91.8%), and two new compounds were obtained. The new compounds were 1,4-O-dicaffeoyl-3-succinyl methyl ester quinic acid (14.6 mg, 96.1%) and 1,5-O-dicaffeoyl-3-O-succinyl methyl ester quinic acid (3.1 mg, 92.6%), respectively. The research indicated that the combination of HSCCC and semi-preparative HPLC is a highly efficient approach for preparative separation of the instability and bioactive caffeoylquinic acid derivatives from natural products.