[Effects of different field processing methods on volatile components of Chuanxiong Rhizoma: an exploration based on headspace gas chromatography-mass spectrometry].

The volatile oil of Chuanxiong Rhizoma(CX) is known as an effective fraction. In order to seek a suitable method for processing CX and its decoction pieces, this study selected 16 volatile components as indices to investigate how different processing methods such as washing/without washing, sun-drying, baking, oven-drying and far-infrared drying at different temperatures affected the quality of CX and its decoction pieces(fresh CX was partially dried, cut into pieces, and then dried) by headspace gas chromatography-mass spectrometry(GC-MS), cluster analysis, principal component analysis and comprehensive weighted scoring. The results showed that the rapid washing before processing did not deteriorate the volatile components of CX. Considering the practical condition of production area, oven-drying was believed to be more suitable than sun-drying, baking, and far-infrared drying. The CX decoction pieces with a thickness of 0.3-0.4 cm were recommended to be oven-dried at 50 ℃. The integrated processing(partial drying, cutting into pieces, and drying) did not cause a significant loss of volatile components. For the fresh CX, the oven-drying at 60 ℃ is preferred. The temperature should not exceed 60 ℃, and drying below 60 ℃ will prolong the processing time, which will produce an unfavorable effect on volatile components. This study has provided the scientific evidence for field processing of CX, which is conducive to realizing the normalization and standardization of CX processing in the production area and stabilizing the quality of CX and its decoction pieces.

[Comparative study on free and bound phenolic acids before and after drying of Salvia miltiorrhiza].

There were significant differences in phenolic acid content between fresh and dried Salvia miltiorrhiza before and after drying. That is to say, the content of phenolic acid in S. miltiorrhiza significantly increased with the increase of dehydration during the drying process.In order to investigate the differences and transformation of free and bound phenolic acids before and after the drying process of S.miltiorrhiza, we studied hydrolysis method, hydrolysates and hydrolysis regularity of phenolic acids in S.miltiorrhiza. UPLC method was used to determine four main hydrolysates of bound phenolic acids, namely danshensu, caffeic acid dimer(SMND-309), caffeic acid, przewalskinic acid A(prolithosperic acid), and three main free phenolic acids in S.miltiorrhiza, namely rosmarinic acid, lithospermic acid, salvianolic acid B. The results of the acid-base hydrolysis experiment of salvianolic acid showed that the alkaline hydrolysis effect was significantly better than acid hydrolysis. The optimal alkaline hydrolysis condition was hydrolysis at 70 ℃ for 4 h with 2 mol·L~(-1) NaOH solution containing 1% ascorbic acid(Vit C). The hydrolysates of free phenolic acids were the same with the hydrolysates of bound phenolic acids. Fresh S.miltiorrhiza contains a low level of free phenolic acids and a high level of bound phenolic acids, which were exactly opposite to dried S.miltiorrhiza. It was suggested that a large amount of bound phenolic acids was accumulated during the growth of S.miltiorrhiza. These bound phenolic acids were coupled with polysaccharides on the cytoderm through ester bonds to form insoluble phenolic acids, which was not easy to be detected by conventional methods. However, during drying and dehydration processes, the bound phenolic acids were converted to a large amount of free phenolic acids under the action of the relevant enzyme.

Comprehensive quality evaluation of the lateral root of Aconitum carmichaelii Debx. (Fuzi): Simultaneous determination of nine alkaloids and chemical fingerprinting coupled with chemometric analysis.

Amino alcohol alkaloids are the active components in the lateral root of Aconitum carmichaelii Debx. (Fuzi), and they have a variety of pharmacological activities. However, the chemical fingerprints of the ester alkaloids reported to date were mainly obtained from high-performance liquid chromatography coupled with ultraviolet detection, and it is difficult to obtain information about amino alcohol alkaloids in Fuzi from such chromatograms. In this paper, a comprehensive fingerprinting method was established using high-performance liquid chromatography coupled with an evaporative light-scattering detector for the simultaneous quantitative analysis of both the amino alcohol alkaloids and ester alkaloids. A total of 42 samples of Fuzi from four production areas were analyzed by constructing high-performance liquid chromatography fingerprints. Then, the quantitative results of the chemical fingerprints combined with chemometrics methods were employed to reveal the factors affecting the geo-authentic Fuzi and to determine characteristic components that can be used to identify these samples. The results indicated distinct differences in the alkaloid contents among samples from the four regions; the geographical origin may be the primary factor affecting the geo-authentic Fuzi, and 15 major components (including songorine, neoline, and hypaconitine, which were quantitatively determined) were found to be characteristic components for the discrimination of Fuzi samples from various regions. Neoline might be a critical component for identifying geo-authentic Fuzi. This approach is convenient, reproducible and provides a promising method for the quality evaluation of Fuzi.

[Study on dynamic changes of chemical constituents during different drying processes of Salvia miltiorrhiza].

There is no consensus on the drying methods of Salvia miltiorrhiza in ancient and modern times,especially on the content of phenolic acid in fresh S. miltiorrhiza. In order to further explore the content of main components in fresh S. miltiorrhiza and study the dynamic changes during the drying process,the content of main components was used as the index in this study to evaluate the processing method,drying method,correlation between dehydration rate and component content for fresh S. miltiorrhiza. In addition,the sealed and unsealed parallel control groups were set to carry out verification test during the drying process. UPLC method was used for determination of seven main components including rosmarinic acid,lithosperic acid,salvianolic acid B,cryptotanshinone,tanshinoneⅠ,methylene salianolate and tanshinone ⅡAin S. miltiorrhiza. The results showed that the fresh S. miltiorrhiza contained low levels of phenolic acid,and the content of phenolic acid increased significantly with the increase of dehydration rate during drying process,while the change of tanshinone was not obvious. In the comparison of three drying methods,we found that drying at 50 ℃ was better than drying in the sun,and drying in the sun was superior to drying in the shade. So,drying at 50 ℃ was the best drying method. The correlation between dehydration and phenolic acid content of S. miltiorrhiza was analyzed by verification test and SPSS software,which further proved that the dehydration rate was significantly positively correlated with the content of phenolic acid components. This study provides reference for the production processing and drying methods of S. miltiorrhiza medicinal materials,which is of great significance for improving the quality of S. miltiorrhiza.

Combinational Treatment of Curcumin and Quercetin against Gastric Cancer MGC-803 Cells in Vitro.

Gastric cancer remains a major health problem worldwide. Natural products, with stronger antitumor activity and fewer side effects, are potential candidates for pharmaceutical development as anticancer agents. In this study, quercetin and curcumin were chosen for testing and were applied separately and in combination to human gastric cancer MGC-803 cells. The MTT assay was used to evaluate cell growth inhibition. Annexin V-FITC/PI was carried out to measure apoptosis rate. Flow cytometry was performed to analyze mitochondrial membrane potential levels. Western blots were applied to detect expression of cytochrome c, total and phosphorylated ERK and AKT. Combined treatment with curcumin and quercetin resulted in significant inhibition of cell proliferation, accompanied by loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c and decreased phosphorylation of AKT and ERK. These results indicate that the combination of curcumin and quercetin induces apoptosis through the mitochondrial pathway. Notably, effect of combined treatment with curcumin and quercetin on gastric cancer MGC-803 cells is stronger than that of individual treatment, indicating that curcumin and quercetin combinations have potential as anti-gastric cancer drugs for further development.

Comparative analysis of diosgenin in Dioscorea species and related medicinal plants by UPLC-DAD-MS.

BACKGROUND: Dioscorea is a genus of flowering plants, and some Dioscorea species are known and used as a source for the steroidal sapogenin diosgenin. To screen potential resource from Dioscorea species and related medicinal plants for diosgenin extraction, a rapid method to compare the contents of diosgenin in various plants is crucial. RESULTS: An ultra-performance liquid chromatography (UPLC) coupled with diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS) method was developed for identification and determination of diosgenin in various plants. A comprehensive validation of the developed method was conducted. Twenty-four batches of plant samples from four Dioscorea species, one Smilax species and two Heterosmilax species were analyzed by using the developed method.The present method presented good sensitivity, precision and accuracy. Diosgenin was found in three Dioscorea species and one Heterosmilax species, namely D. zingiberensis, D. septemloba, D. collettii and H. yunnanensis. CONCLUSION: The method is suitable for the screening of diosgenin resources from plants. D. zingiberensis is an important resource for diosgenin harvesting.

Chemical profile analysis and comparison of two versions of the classic TCM formula Danggui Buxue Tang by HPLC-DAD-ESI-IT-TOF-MSn.

Danggui Buxue Tang (DBT) is a Traditional Chinese Medicine (TCM) formula primarily used to treat symptoms associated with menopause in women. Usually, DBT is composed of one portion of Radix Angelicae Sinensis (RAS) and five portions of Radix Astragali (RA). Clinically, Radix Hedysari (RH) is sometimes used by TCM physicians to replace RA in DBT. In order to verity whether the chemical constituents of the DBT1 (RA:RAS = 5:1, w/w) and DBT2 (RH:RAS = 5:1, w/w) share similarities the chemical profiles of the two DBTs crude extracts and urine samples were analyzed and compared with the aid of HPLC-DAD-ESI-IT-TOF-MSn, which determines the total ion chromatogram (TIC) and multi-stage mass spectra (MSn). Then, the DBT1 and DBT2 were identified and compared on the basis of the TIC and the MSn. In the first experiment (with crude extracts), 69 compounds (C1-C69) were identified from the DBT1; 46 compounds (c1-c46) were identified from the DBT2. In the second experiment(with urine samples), 44 compounds (M1-M44) were identified from the urine samples of rats that had been administered DBT1, and 34 compounds (m1-m34) were identified from the urine samples of rats that had been administered DBT2. Identification and comparison of the chemical compositions were carried out between the DBT1 and DBT2 of the crude extracts and urine samples respectively. Our results showed that the two crude extracts of the DBTs have quite different chemical profiles. The reasons for their differences were that the special astragalosides in DBT1 and the isoflavonoid glycosides formed the malonic acid esters undergo single esterification and acetyl esters undergo acetylation in DBT1. In contrast, the urine from DBT1-treated rats strongly resembled that of DBT2-treated rats. These metabolites originate mainly from formononetin, calycosin and their related glycosides, and they were formed mainly by the metabolic process of reduction, deglycosylation, demethylation, hydrogenation and sulfation. The HPLC-DAD-ESI-IT-TOF-MSn method was successfully applied for the rapid chemical profiles evaluation of two DBTs and their related urine samples.

Quantitative comparison of multiple components in Dioscorea nipponica and D. panthaica by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Dioscorea nipponica (DN) and D. panthaica (DP) have been uniquely prepared as herbal medicinal products for treating coronary heart disease (CHD) in China. However, so far there has been little discussion and no work comparing the qualitative and quantitative differences between the two herbs nor assessing whether they have similarities in chemical composition that would support their common application for treating CHD. OBJECTIVE: To develop an efficient and reliable method based on UPLC-qTOF-MS for quantitative comparison of saponins in both DN and DP. METHODS: Using electrospray ionisation and atmospheric-pressure chemical ionisation respectively, six steroidal glycosides and one aglycone were determined in 13 DN samples and 13 DP samples. The comparative analysis of chemical components in DN and DP was carried out by chromatographic fingerprint similarity evaluation, test of significance (t-test) and principle component analysis (PCA). RESULTS: The UPLC-qTOF-MS method showed limit of detection and quantitation within the range 0.02-0.2 ng and 0.08-0.5 ng, respectively, for the seven saponins studied. The intra- and interday precision (RSD) were below 5%. The recoveries for the quantified compounds were within the range of 72.79-118.31%. CONCLUSION: This UPLC-qTOF-MS assay provides a suitable method for the identification and determination of major bioactive constituents both in DN and DP. The chemical composition of all DN and DP samples studied exhibited a high level of global similarity. This chemical similarity validates their common application in the pharmaceutical industry as anti-CHD herbal drugs.

Apoptosis sensitization by Euphorbia factor L1 in ABCB1-mediated multidrug resistant K562/ADR cells.

In this article, reversal activities of Euphorbia factor L1 (EFL1) against ABCB1-mediated multidrug resistance (MDR) and apoptosis sensitization in K562/ADR cells are reported. EFL1 decreased the IC50 values of anticancer agents in K562/ADR cells over-expressing ABCB1. However, EFL1 did not affect the IC50 values of anticancer agents in sensitive K562 cells. Additionally, EFL1 increased the intracellular accumulation of rhodamine 123 and doxorubicin in K562/ADR cells without affecting their accumulation in K562 cells. Furthermore, EFL1 sensitized the apoptosis triggered by vincristine in K562/ADR cells via mitochondrial pathway, as confirmed by Annexin V-FITC/PI detection and western blot. At the same time, EFL1 did not influence the apoptosis induced by vincristine in K562 cells. Western blot results showed that EFL1 did not affect the phosphorylation level of AKT and ERK in K562 and K562/ADR cells. Finally, EFL1 did not down-regulate protein expression of ABCB1.

Profiling complex volatile components by HS-GC-MS and entropy minimization software: An example on Ligusticum chuanxiong Hort.

Volatile oil, as an important bioactive fraction of medicinal herbs, is comprised of a diversity of compounds. At present, gas chromatography-mass spectrometry (GC-MS) is one of the mainstream approaches to profiling these complex components. However, GC-MS faces the major bottleneck in data analysis, such as co-elution of more than one compound, and interference caused by high background noise; this usually makes an operator have to spend a lot of time and effort in optimizing experimental conditions. Taking Chuanxiong Rhizoma (the dry rhizome of Ligusticum chuanxiong Hort., abbreviated as "CR") as an example, this study is intended to provide a feasible, quick and cost-effective solution for compound identification based on the chemometric method of entropy minimization (EM) algorithm. Ten batches of geo-authentic CR and eight batches of adulterants including Fuxiong (FX), Shanchuanxiong (SCX) and Cnidii Rhizoma (CNR) were determined by headspace GC-MS. FX and SCX were rhizomes of L. chuanxiong but subjected to improper harvest time. CNR was the dried rhizome of Cnidium officinale Makino. The co-eluting and overlapping peaks and low-concentration peaks with high background were precisely reconstructed by EM algorithm, and then the reconstructed pure mass spectra of each component were compared with the ion fragment information in NIST library for qualitative identification. EM algorithm proves to be capable of delivering results with increased accuracy and high confidence. Moreover, by the GC-MS approach established in this work, the volatile chemical profiles of FX, SCX, and CNR, were quite distinct from those of geo-authentic CR, suggesting that the adulterants should not be confused with CR in clinical practice and pharmaceutical industry. In brief, the advanced EM algorithm is envisioned to be applied to a variety of medicinal herbs, enabling rapid and accurate identification of volatile phytochemicals.

Determination of Inorganic Elements in the Rhizome of Paris polyphylla Smith Var. chinensis (Franch.) Hara by Using Inductively Coupled Plasma Mass Spectrometry.

The objective of this study was to investigate the concentrations of inorganic elements in the rhizome of Paris polyphylla Smith var. chinensis (Franch.) Hara of different planting years and cultivation conditions. Twenty-five inorganic elements including Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, P, Pb, Se, Sr, Ti, V, and Zn in the rhizome were determined by using inductively coupled plasma mass spectrometry (ICP-MS). The analytical method was validated by measuring several parameters including linearity, correlation coefficient, limit of detection (LOD), limit of quantification (LOQ), and recovery. The linear working ranges were three, 0-300 µg/L, 0-500 µg/L, and 0-1000 µg/L, and the correlation coefficients (r) values were higher than 0.998. LOD varied from 0.001 µg/L (Be) to 11.957 µg/L (P), and LOQ varied from 0.003 µg/L (Be) to 35.870 µg/L (P). The recoveries spanned from 95.2 (Co) to 105.3% (Pb). Validation parameters showed the possibility of using whole of the sample preparation procedures used in this study. Based on the determined results, it is indicated that the toxic elements As, Cd, and Pb had no ingestion risk. The planting years and cultivation conditions had significant effects on the concentrations of inorganic elements of P. polyphylla var. chinensis. The concentrations of inorganic elements in cultivated samples were different from those in wild samples. The results can provide useful theoretical basis for the quality control and rational use of P. polyphylla var. chinensis.

Preparation-related structural diversity and medical potential in the treatment of diabetes mellitus with ginseng pectins.

Pectins isolated from Panax ginseng C.A. Meyer are potential therapeutic agents for the treatment of diabetes mellitus, a global health challenge. Soil-to-bench procedures of ginseng pectin preparation significantly affect the polysaccharide structures. Various forms of ginseng pectins rich in homogalacturonan, rhamnogalacturonan-I, rhamnogalacturonan-II, and arabinogalactan have demonstrated independent or collaborative effects on hyperglycemia, oxidative stress, immunological dysfunction, and neoplasms. Monosaccharide compositions, peptide contents, degrees of esterification and methylation, and inter- and intramolecular linkages all influence pectin bioactivity. Understanding the preparation-structure and structure-function relationships of ginseng pectins can lead to safer and more pertinent treatment of diabetes with efficacy-oriented modifications of the pectins. To reach this goal, standardization of preparation procedures, understanding of intricate structures, and exploration of complex interactions with receptors are crucial steps to take full advantage of the medical potential of ginseng pectins.

Biotransformation of Dioscorea nipponica by Rat Intestinal Microflora and Cardioprotective Effects of Diosgenin.

Studying the biotransformation of natural products by intestinal microflora is an important approach to understanding how and why some medicines-particularly natural medicines-work. In many cases, the active components are generated by metabolic activation. This is critical for drug research and development. As a means to explore the therapeutic mechanism of Dioscorea nipponica (DN), a medicinal plant used to treat myocardial ischemia (MI), metabolites generated by intestinal microflora from DN were identified, and the cardioprotective efficacy of these metabolites was evaluated. Our results demonstrate that diosgenin is the main metabolite produced by rat intestinal microflora from DN. Further, our results show that diosgenin protects the myocardium against ischemic insult through increasing enzymatic and nonenzymatic antioxidant levels in vivo and by decreasing oxidative stress damage. These mechanisms explain the clinical efficacy of DN as an anti-MI drug.

Comparative evaluation of chemical profiles of three representative 'snow lotus' herbs by UPLC-DAD-QTOF-MS combined with principal component and hierarchical cluster analyses.

Herbal healthcare products are used worldwide as relatively safe and effective alternatives to allopathic drugs. Saussurea laniceps Hand.-Mazz. (SL), S. medusa Maxim. (SM) and S. involucrata (Kar. et Kir.) Sch.Bip. (SI) are three sources of the renowned 'snow lotus', Chinese materia medica for treating inflammatory diseases. The three species have different therapeutic effects, among which SL has been proved to be the most potent, but they are frequently confused on the market and in the academic community. An ultra-high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry (UPLC-DAD-QTOF-MS) method was developed and used to analyze 49 herbal samples for species analysis and overall quality evaluation. With 25 simultaneously identified constituents, of which 12 were quantified, the three herbs showed different chemical profiles. Four-dimensional principle component analysis (4D-PCA) and orthogonal hierarchical cluster analysis (2D-HCA) results illustrated that SL should be grouped away from SM and SI, contradicting the botanical record in Flora of China. The present chemical determination and pattern recognition results directly explain the therapeutic potency of SL and distinguish the three confused snow lotus herbs. Furthermore, the findings suggest a possible extensive quality evaluation model for multi-origin medicinal plants and help monitor falsification of snow lotus herbal products on the market, contributing to a more regulated pharmaceutical industry. Copyright © 2016 John Wiley & Sons, Ltd.

Assessing the quality of Smilacis Glabrae Rhizoma (Tufuling) by colormetrics and UPLC-Q-TOF-MS.

BACKGROUND: The quality of the materials used in Chinese medicine (CM) is generally assessed based on an analysis of their chemical components (e.g., chromatographic fingerprint analysis). However, there is a growing interest in the use of color metrics as an indicator of quality in CM. The aim of this study was to investigate the accuracy and feasibility of using color metrics and chemical fingerprint analysis to determine the quality of Smilacis Glabrae Rhizoma (Tufuling) (SGR). The SGR samples were divided into two categories based on their cross-sectional coloration, including red SGR (R-SGR) and white SGR (W-SGR). METHODS: Forty-three samples of SGR were collected and their colors were quantized based on an RGB color model using the Photoshop software. An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) system was used for chromatographic fingerprint analysis to evaluate the quality of the different SGR samples. Hierarchical cluster analysis and dimensional reduction were used to evaluate the data generated from the different samples. Pearson correlation coefficient was used to evaluate the relationship between the color metrics and the chemical compositions of R-SGR and W-SGR. RESULTS: The SGR samples were divided into two different groups based on their cross-sectional color, including color A (CLA) and B (CLB), as well as being into two separate classes based on their chemical composition, including chemical A (CHA) and B (CHB). Standard fingerprint chromatograms were for CHA and CHB. Statistical analysis revealed a significant correlation (Pearson's r = -0.769, P < 0.001) between the color metrics and the results of the chemical fingerprint analysis. CONCLUSIONS: The SGR samples were divided into two major clusters, and the variations in the colors of these samples reflected differences in the quality of the SGR material. Furthermore, we observed a statistically significant correlation between the color metrics and the quality of the SGR material.

HSCCC-based strategy for preparative separation of in vivo metabolites after administration of an herbal medicine: Saussurea laniceps, a case study.

This paper reports a novel strategy based on high-speed counter-current chromatography (HSCCC) technique to separate in vivo metabolites from refined extract of urine after administration of an herbal medicine. Saussurea laniceps (SL) was chosen as a model herbal medicine to be used to test the feasibility of our proposed strategy. This strategy succeeded in the case of separating four in vivo metabolites of SL from the urine of rats. Briefly, after oral administration of SL extract to three rats for ten days (2.0 g/kg/d), 269.1 mg of umbelliferone glucuronide (M1, purity, 92.5%), 432.5 mg of scopoletin glucuronide (M2, purity, 93.2%), 221.4 mg of scopoletin glucuronide (M3, purity, 92.9%) and 319.0 mg of scopoletin glucuronide (M4, purity, 90.4%) were separated from 420 mL of the rat urine by HSCCC using a two-phase solvent system composed of methyl tert-butyl ether-n-butanol-acetonitrile-water (MTBE-n-BuOH-ACN-H2O) at a volume ratio of 10:30:11:49. The chemical structures of the four metabolites, M1 to M4, were confirmed by MS and (1)H, (13)C NMR. As far as we know, this is the first report of the successful separation of in vivo metabolites by HSCCC after administration of an herbal medicine.

Bruceine D induces apoptosis in human chronic myeloid leukemia K562 cells via mitochondrial pathway.

Chronic myeloid leukemia (CML), an acquired malignant myeloproliferative disorder of hematopoietic stem cells, is one of the three most common forms of leukemia. In this study, we investigated the effects of bruceine D, which have been isolated from Brucea javanica (L.) Merr. on human chronic myeloid leukemia K562 cells. MTT assay was used to evaluate cell growth inhibition. Flow cytometry was performed to analyze mitochondrial membrane potential (ΔΨm). Western blot was applied to detect expression of cytochrome c, caspases-9, -3, PARP and other proteins. Bruceine D exhibited potent cytotoxicity to K562 cells with IC50 of 6.37 ± 0.39 µM. It led to loss of ΔΨm, release of cytochrome c, activation of caspases-9, -3 and cleavage of PARP, which suggested that bruceine D induced apoptosis of K562 cells through mitochondrial pathway. In addition, bruceine D inhibited the phosphorylation of AKT and ERK. It's indicative that the potent anticancer activity of bruceine D be related to MAPK and PI3K pathways.

The variation in the major constituents of the dried rhizome of Ligusticum chuanxiong (Chuanxiong) after herbal processing.

BACKGROUND: Rhizoma Chuanxiong (RC; Chuanxiong), which is the dried rhizome of Ligusticum chuanxiong (Umbelliferae), is commonly used in Chinese medicine (CM) for improving blood circulation and dispersing blood stasis. RC is usually processed before use in clinical practice to enhance its therapeutic efficacy. This study aimed to investigate the temporal variations of the major constituents of RC by HPLC-DAD-MS during herbal processing to investigate the effects of an adjuvant (e.g., wine), steaming vs stir-frying and the optimal processing time. METHODS: An HPLC-DAD-MS method was developed to determine the major constituents of the RC processed by one of the four processing methods, i.e., stir-frying, steaming, stir-frying with rice wine and steaming with rice wine. Processing was conducted over 60 min. Six major compounds, namely ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A, Z-ligustilide and levistolide A, were selected as markers to analyze the effects on the markers' levels of the different processing methods and optimize the processing time. RESULTS: The results indicated that (a) processing with wine had no discernible impact on the amounts of the six chemical markers in RC; (b) the amounts of the major constituents of RC subjected to steam processing were higher than those of the RC subjected to stir-fry processing. CONCLUSION: Among the four different methods evaluated for RC processing, steaming was better and the optimal time for steaming RC was 40 min.

Cardioprotective effect of total saponins from three medicinal species of Dioscorea against isoprenaline-induced myocardial ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: As folk medicines used in China since 1950s, Dioscorea nipponica Makino (DN), D. panthaica Prain et Burkill (DP), and D. zingiberensis C.H. Wright (DZ) are regarded as having more or less the same traditional therapeutic actions, such as activating blood, relieving pain, and dispersing swelling. It is noteworthy that, of the 49 species of the genus Dioscorea distributed in China, based on such traditional efficacies, only these three have been further developed as effective single-herb medicines for treating cardiovascular diseases by the modern pharmaceutical industry. In our previous study, it was found that the chemical compositions of DN and DP were similar, and both were distinct from that of DZ. Hence, whether their different chemical profiles support their anti-IHD (ischemic heart disease) activity in common still needs to be answered. So far it is still unknown whether the efficacies of these three herbs act via similar mechanism and whether they possess comparable therapeutic efficacy for experimental myocardial ischemia (MI). AIM OF THE STUDY: The present study aimed to further investigate the underlying mechanisms with respect to antioxidative stress activity by which these Dioscorea spp. attenuate MI, and to compare the therapeutic effect of total saponins from these three species on myocardial antioxidant levels and myocardial histology. MATERIAL AND METHODS: The serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), total superoxide dismutases (SOD), catalase (CAT), glutathione peroxidase (GPx), the total antioxidant capacity (T-AOC), and malondialdehyde (MDA), as well as myocardial histology, were compared among rat groups administered with total saponins (TS) of DN, DP or DZ (abbreviated as DNTS, DPTS and DZTS, respectively). The rats experienced myocardial ischemia induced by isoprenaline (ISO) injection; the test solutions (DNTS, DPTS, DZTS) were administered either after the ISO injection, or both before and after. RESULTS: Compared with the model group (ISO injection only), TS groups exhibited significantly reduced activities of CK, LDH and AST, lowered level of MDA, and increased activities of SOD, CAT, GPx and T-AOC; heart tissues from TS groups revealed less severe histological damage. The cardioprotective efficacy of these three Dioscorea TS for rat MI was closely comparable based on the above observations. CONCLUSION: The findings of the present study provide evidence that the anti-MI effect of DNTS, DPTS and DZTS can be attributed to the increase of myocardial antioxidant levels and decrease of lipid peroxidation formation, and the closely comparable results observed from these three Dioscorea saponins thereby explains the similarity in their clinical efficacy as anti-MI drugs.

UPLC-QTOF-MS identification of metabolites in rat biosamples after oral administration of Dioscorea saponins: a comparative study.

ETHNOPHARMACOLOGICAL RELEVANCE: Among the 49 species of the genus Dioscorea distributed in China, Dioscorea nipponica Makino (DN), Dioscorea panthaica Prain et Burkill (DP), and Dioscorea zingiberensis C. H. Wright (DZ) possess more or less similar traditional therapeutic actions, such as activating blood, relieving pain, and dispersing swelling; they have been used as folk medicine in China since 1950s. The modern pharmaceutical industry has developed these three species as herbal medicines that have been used for decades for treating cardiovascular diseases. However, there is no available information in the literature explaining how their chemical components are converted and interrelated in vivo to support their efficacies. The present study aimed to a) compare the metabolic profiles of saponins from DN, DP and DZ, which are considered to be their bioactive components, and b) to compare the changes in sustained levels of metabolites from rat biosamples. MATERIAL AND METHODS: Total saponins (TS) from each of the three species, and four individual saponins, namely protodioscin (PD), pseudoprotodioscin (PSD), dioscin (DC) and diosgenin (DG), were given to rats by oral administration. Chemical profiles of the rats' plasma, urine and feces were monitored 1-36 h. A UPLC-QTOF-MS based method was performed to identify the absorbed constituents and their metabolic products in rat biosamples (i.e., blood, urine, and feces); the ratio of peak area of major saponins to that of internal standard was calculated and plotted versus time to characterize the sustained levels of saponins in biosamples. RESULTS: Totally 10 saponin-related compounds were detected in rat plasma, 10 in rat urine and 18 in rat feces. The results indicated that formation of diosgenin by desugarization was the main pathway by which steroidal glycosides were metabolized. Other types of bio-transformation were found among glycosides and aglycones, such as ring cyclization through loss of 26-O-glucosyl, substitution of ß-D-glucopyranosyl for α-L-rhamnopyrannosyl, hydrogenation of diosgenin at 5(6)-double bond, and hydration of 20(22)-double bond. Generally, the metabolic profiles of DN and DP were shown to be quite similar, but different from that of DZ. However, some particular similarities and connections were found among these three TS. Diosgenin was one of the main metabolites commonly found in plasma and feces (excluding urine), from all groups receiving different TS, as well as individual saponins; this is likely to be one of the bioactive constituents playing an essential role in cardioprotective efficacy. Furostane-type saponins in TS of DN, DP or DZ, such as PD, protogracillin, parvifloside, protodeltonin and protobioside, showed fast absorption into blood (<1h), but were maintained for a relatively short period (mostly<8h), while the spirostane-type saponin and sapogenin (DC and DG, respectively), were absorbed into circulation more slowly (>1h), but increased gradually and lasted longer (>36h). These two patterns suggest that the therapeutic effect of these Dioscorea saponins is achieved through a complex, multi-step process over time. In addition, it appears that PD, PSD, and DC contained in DN and DP were transformed into certain glycosides originally found in DZ but not in DN or DP (protodeltonin, deltonin, trillin, and progenin II), which might indicate another linkage among these three species. CONCLUSION: These similarities and connections described above constitute evidence supporting similarity in efficacy of these three herbs from the perspective of metabolism. The UPLC-QTOF-MS based method is accurate and efficient for analyzing metabolic changes in rat biosamples over time.