Application of smartphone in detection of thin-layer chromatography: Case of salvia miltiorrhiza.

In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components of Salvia miltiorrhiza. The key construction and shooting parameters were investigated by the relative peak area and signal-to-noise ratio. The best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58°; exposure compensation, 0~0.2 EV; and shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi-quantitative analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S. miltiorrhiza. The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e., Gelanalyzer and ImageJ. Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.

Synergistic immunomodulatory effect of complex polysaccharides from seven herbs and their major active fractions.

In this report, we present the strategy for the revelation of synergistic effect and elucidation of active fractions from an immunomodulatory complex polysaccharide derived from seven herbs (Lentinula edodes, Ganodorma lucidum, Tremella fuciformis, Chrysanthemum, Lycium barbarum, Codonopsis pilosula and Poria cocos), a formula used as health product in China market, using the combination of HPSEC-MALLS, immunological bioassay and saccharide mapping analysis. The effects of complex polysaccharide and their fractions on RAW 246.7 macrophages demonstrated that the fractions (CD1, CD2, CD3) with molecular weight above 10 kDa exhibited immune activity by directly stimulated NO release and phagocytosis, and induced macrophages to secrete cytokines. Especially, fraction CD2 with molecular weight of 100-1000 kDa showed the strongest bioactivity (EC50 = 0.19 µg/mL) compared with their individual corresponding herbal polysaccharides fractions due to synergistic effect, which supported the scientific use of Chinese herbal mixture. Moreover, their chemical characters were analyzed by HPSEC-MALLS and saccharide mapping, and the original herbs, including L. edodes, G. lucidum, T. fuciformis and Chrysanthemum, responsible for the immunomodulatory activity were tentatively revealed. Results are beneficial for the quality analysis and formula optimization of complex polysaccharides in both biomedical and functional food field.

Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice.

BACKGROUND: Shexiang Baoxin Pill (SBP), a formulated traditional Chinese medicine (TCM), has been widely used to treat cardiovascular diseases for years. This herbal mixture has been shown to promote differentiation of cultured neuronal cells. Here, we aimed to investigate the effects of SBP in attenuating cognitive impairment in APP/PS1 transgenic mice. METHODS: Ethanol and water extracts of SBP, denoted as SBPEtOH and SBPwater, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBPEtOH extract in attenuating the cognitive impairments in APP/PS1 transgenic mice was shown by following lines of evidence: (i) inhibition of Aß fibril formation, (ii) suppression of secretions of cytokines, and (iii) improvement of behavioral tests by Morris water maze. RESULTS: SBPwater and SBPEtOH inhibited the formation of ß-amyloid fibrils and protected the Aß-induced cytotoxicity in cultured PC12 cells. In APP/PS1 transgenic mice, the treatment of SBPEtOH inhibited expressions of NO, NOS, AChE, as well as aggregation of Aß. Besides, the levels of pro-inflammatory cytokines were suppressed by SBP treatment in the transgenic mice. Importantly, the behavioral tests by Morris Water maze indicated that SBP attenuated cognitive impairments in APP/PS1 transgenic mice. CONCLUSION: The current result has supported the notion that SPB might ameliorate the cognitive impairment through multiple targets, suggesting that SBP could be considered as a promising anti-AD agent.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish.

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

Effects of extraction methods on immunology activity and chemical profiles of Lycium barbarum polysaccharides.

It has been proven that polysaccharides have bioactivities and are beneficial to cure many diseases. Lycium barbarum fruit is widely used as a functional food all over the world, which main active component is L. barbarum polysaccharides (LBPs). In this study, classical hot water extraction (HWE), microwave assisted extraction (MAE), ultrasonic assisted extraction (UAE) and pressurized liquid extraction (PLE) were used to extracted LBP. The chemical properties of LBPs were evaluated in terms of total polysaccharide contents, uronic acid contents and protein contents. High performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector was applied to measure the characters such as molecular weight, radius of gyration and polydispersity index. Then the immunomodulatory activity of LBPs was evaluated through RAW 264.7 cells. The results showed that HWE was the best method to get the highest total sugar and acidic polysaccharides, MAE was preferable to extract polysaccharide-protein complex, but PLE, UAE and HWE could get better immunomodulatory activity polysaccharides than MAE. Besides, the peak 3 in chromatogram of MAE extracted LBPs was obviously higher than those of LBPs produced by other 3 extraction methods, which suggested that peak 1 and peak 2 might be biologically active polysaccharides fractions in LBPs. Therefore, effect of different extraction methods on structure and composition of LBPs attributed to their variance of immunological activities.

Simultaneous extraction, separation, isolation and identification of endogenous components from Etlingera elatior by pressurized matrix solid-phase dispersion using liquid chromatography-mass spectrometry.

From the analytical chemistry point-of-view, an ideal sample preparation method should be simple, rapid, automatic, selective, precise, exhaustive, reproducible and protect the analyte-of-interest from degradation. In this study, a novel sample preparation method, named pressurized matrix solid-phase dispersion (p-MSPD) extraction was developed for simultaneously extracting, separating, purifying, isolating, and analyzing endogenous components in a solid sample matrix. Etlingera elatior, a traditional medicinal plant known as the torch ginger, was applied as a sample matrix to evaluate the p-MSPD process. The entire extraction, separation, isolation, fractionation and detection were performed automatically with a commercial LC-MS system. The novel method was satisfactorily applied for the preparation of real samples without optimization, which had the ability to selectively isolate pure compounds from the solid sample matrix for further NMR analysis. Therefore, the method is recommended for quality control of traditional medicines, research efforts when sample amounts are limited, and laboratories that have ordinary LC-MS instrumentation.

[Study on quantification method of non-starch polysaccharides in Dendrobium catenatum].

The non-starch polysaccharides,mainly composed of glucomannans,are the major bioactive compounds in Dendrobium catenatum. In order to evaluate the quality of the medicinal materials and guide the production and processing,a quantification method of non-starch polysaccharides was established by stems of D. catenatum C15 strain collected from the pear epiphytic cultivation. The non-starch polysaccharides were obtained by " water extraction,α-amylase pretreatment,and alcohol precipitation once" method. The contents of starches,non-starch polysaccharides and monosaccharides were analyzed. In addition,the system suitability was tested. Compared with method of the Chinese Pharmacopoeia( 2015 edition),the contents of total polysaccharides,glucose,and mannose were decreased by 20. 9%,58. 8% and 1. 6% respectively. The method effectively digested starch and retained non-starch polysaccharides,and the analysis result was accurate and repeatable. Therefore,it is suitable for the content measurement of non-starch polysaccharides of D. catenatum. Furthermore,it could be an alternative method for quality control of D. catenatum and a reference in the determination of non-starch polysaccharides in other starch-containing medicinal materials.

Recent applications of immobilized biomaterials in herbal analysis.

Immobilization of biomaterials developed rapidly due to the great promise in improving their stability, activity and even selectivity. In this review, the immobilization strategies of biomaterials, including physical adsorption, encapsulation, covalent attachment, cross-linking and affinity linkage, were briefly introduced. Then, the major emphasis was focused on the reported various types of immobilized biomaterials, including proteins, enzymes, cell membrane and artificial membrane, living cells, carbohydrates and bacteria, used in the herbal analysis for bioactive compound screening, drug-target interaction evaluation and chiral separation. In addition, a series of carrier materials applied in biomaterials immobilization, such as magnetic nanoparticles, metal-organic frameworks, silica capillary column, cellulose filter paper, cell membrane chromatography, immobilized artificial membrane chromatography and hollow fiber, were also discussed. Perspectives on further applications of immobilized biomaterials in herbal analysis were finally presented.

Simultaneous Quantification of Three Curcuminoids and Three Volatile Components of Curcuma longa Using Pressurized Liquid Extraction and High-Performance Liquid Chromatography.

A high-performance liquid chromatography (HPLC) method was investigated for the simultaneous quantification of two chemical types of bioactive compounds in the rhizome of Curcuma longa Linn. (turmeric), including three curcuminoids: Curcumin, bisdemethoxycurcumin, and demethoxycurcumin; and three volatile components: ar-turmerone, ß-turmerone, and α-turmerone. In the present study, the sample extraction system was optimized by a pressurized liquid extraction (PLE) process for further HPLC analysis. The established HPLC analysis conditions were achieved using a Zorbax SB-C18 column (250 mm × 4.6 mm i.d., 5 µm) and a gradient mobile phase comprised of acetonitrile and 0.4% (v/v) aqueous acetic acid with an eluting rate of 1.0 mL/min. The curcuminoids and volatile components were detected at 430 nm and 240 nm, respectively. Moreover, the method was validated in terms of linearity, sensitivity, precision, stability and accuracy. The validated method was successfully applied to evaluate the quality of twelve commercial turmeric samples.

Preparation and Application of Standardized Typical Volatile Components Fraction from Turmeric (Curcuma longa L.) by Supercritical Fluid Extraction and Step Molecular Distillation.

A green and reliable method using supercritical fluid extraction (SFE) and molecular distillation (MD) was optimized for the separation and purification of standardized typical volatile components fraction (STVCF) from turmeric to solve the shortage of reference compounds in quality control (QC) of volatile components. A high quality essential oil with 76.0% typical components of turmeric was extracted by SFE. A sequential distillation strategy was performed by MD. The total recovery and purity of prepared STVCF were 97.3% and 90.3%, respectively. Additionally, a strategy, i.e., STVCF-based qualification and quantitative evaluation of major bioactive analytes by multiple calibrated components, was proposed to easily and effectively control the quality of turmeric. Compared with the individual calibration curve method, the STVCF-based quantification method was demonstrated to be credible and was effectively adapted for solving the shortage of reference volatile compounds and improving the QC of typical volatile components in turmeric, especially its functional products.

Advanced strategies for quality control of Chinese medicines.

Quality control is always the critical issue for Chinese medicines (CMs) with their worldwide increasing use. Different from western medicine, CMs are usually considered that multiple constituents are responsible for the therapeutic effects. Therefore, quality control of CMs is a challenge. In 2011, the strategies for quantification, related to the markers, reference compounds and approaches, in quality control of CMs were reviewed (Li, et al., J. Pharm. Biomed. Anal., 2011, 55, 802-809). Since then, some new strategies have been proposed in these fields. Therefore, the review on the strategies for quality control of CMs should be updated to improve the safety and efficacy of CMs. Herein, novel strategies related to quality marker discovery, reference compound development and advanced approaches (focused on glyco-analysis) for quality control, during 2011-2016, were summarized and discussed.

A Novel Strategy for Quantitative Analysis of Major Ginsenosides in Panacis Japonici Rhizoma with a Standardized Reference Fraction.

Panacis Japonici Rhizoma (Zhu-Jie-Shen in Chinese), the root of P. japonicus C.A. Mey., is commonly used in traditional Chinese Medicine. Saponins are the major bioactive compounds in this herb. The similarity of polarity and structure of the natural products in herb caused the difficulty of purification and resulted in the shortage and high cost of the reference compounds, which has greatly hindered efforts toward quantification in quality control. A novel strategy using a standardized reference fraction for qualification of the major saponins in Panacis Japonici Rhizoma was proposed to easily and effectively control the quality of PJR. The strategy is feasible and reliable, and the methodology of the developed approach is also validated. The standardized reference fraction was used for quantification, which might solve the shortage of the pure reference compounds in the quality control of herbal medicines.

[Ganoderma triterpenoids from aqueous extract of Ganoderma lucidum].

A new triterpenoid and 18 analogues were isolated from the water extract of Ganoderma lucidum by column chromatographic techniques, including silica gel, ODS, Sephadex LH-20, and HPLC. The new compound was elucidated as 2ß-acetoxy-3ß,25-dihydroxy-7,11,15-trioxo-lanost-8-en-26-oic acid on the basis of analyses of extensive spectroscopic data and its physicochemical properties. Comparison of NMR data with those reported in literature, the known analogues were determined as ganoderic acid H (2), 12ß-acetoxy-3ß,7ß-dihydroxy-11,15,23-trioxo-lanost-8,16-dien-26-oic acid (3), ganoderenic acid D (4),ganoderic acid C1 (5),ganoderic acid G (6),3ß,7ß-dihydroxy-11,15,23-trioxo-lanost-8,16-dien-26-oic acid (7),ganoderic acid B (8),ganoderic acid C6 (9),3ß,15α-dihydroxy-7,11,23-trioxo-lanost-8,16-dien-26-oic acid (10),ganoderic acid A (11),ganolucidic acid A (12),lucidenic acid E2 (13),lucidenic acid N (14),lucidenic acid P (15), lucidenic acid B (16),lucidenic acid A (17),lucidenic acid C (18),and lucidenic acid L (19), respectively. Compound 1 is new compound and compounds 2-19 have been reported from G. lucidum. The present study enriches the knowledge of the chemical constituent of G. lucidum and completes chemical investigation of water decoction that is traditional use of G. lucidum.

Pharmacokinetic Comparison of Seven Major Bio-Active Components in Normal and Blood Stasis Rats after Oral Administration of Herb Pair Danggui-Honghua by UPLC-TQ/MS.

The compatibility between Danggui (Angelicae Sinensis Radix) and Honghua (Carthami Flos) is a known herb pair, which could activate blood circulation and dissipate blood stasis effects. In this paper, we quantified seven main bio-active components (hydroxysafflor yellow A, caffeic acid, p-coumaric acid, kaempferol-3-O-rutinoside, ferulic acid, 3-n-butylphthalide, and ligustilide) in plasma samples in vivo by UPLC-TQ/MS method and investigatedwhether the pharmacokinetic (PK) behaviors of the seven components could be altered in blood stasis rats after oral administration of the Gui-Hong extracts. It was found that the Cmax and AUC0-t of these components in blood stasis rats had increasing tendency compared with normal rats. Most components in model and normal rats had significant difference in some pharmacokinetic parameters, which indicated that the metabolism enzymes and transporters involved in the metabolism and disposition of these bio-active componentsmay bealtered in blood stasis rats. This study was the first report about the pharmacokinetic investigation between normal and blood stasis rats after oral administrationof Gui-Hong extracts, and these results are important and valuable for better clinical applications of Gui-Hong herb pair and relatedTCM formulae.

Effects of Polysaccharides in Lycium Barbarum Berries from Different Regions of China on Macrophages Function and their Correlation to the Glycosidic Linkages.

Berries of Lycium barbarum L. are not only used for traditional Chinese medicine, but also for raw materials in many health foods. Polysaccharides are major components of L. barbarum berries, which possess a variety of biological activities. In this study, effects of water-soluble polysaccharides, in 8 typical batches of L. barbarum berries collected from different producing areas of China, on macrophage function were evaluated in vitro. Furthermore, to better understand the structure-activity relationship of the polysaccharides in L. barbarum berries, the activity of typical polysaccharides and their partial acid and enzymatic hydrolysates were also investigated and compared. The results showed that the effects of polysaccharides of different regions are similar, which should be correlated to their similar chemical properties. However, their promotion effects on macrophage function are different in degree, this might be caused by their different content of active polysaccharides. Moreover, the α-1,4-d-galactosiduronic and α-1,5-arabinosidic linkages, especially the former one was discovered to significantly affect the promotion effect on macrophage function induced by the polysaccharides in L. barbarum berries. These results were beneficial to improve the pharmacological activity-based quality control of polysaccharides in L. barbarum berries and their products. PRACTICAL APPLICATION: The results showed that immunomodulation effects of polysaccharides in L. barbarum berries (LBPs) from different regions are similar, but different in degree, this might be caused by their different content of bioactive polysaccharides. Moreover, an enzymatic digestion method was used to investigate the structure-bioactivity relationship of polysaccharides from LBPs. The result indicated that α-1,4-d-galactosiduronic and α-1,5-arabinosidic linkages, especially the former one was significantly affect the immunomodulation effects of LBPs. The results were beneficial to the improvement of pharmacological activity-based quality control of LBPs and future development of related unique functional and health products.

Evaluation on quality consistency of Ganoderma lucidum dietary supplements collected in the United States.

Ganoderma lucidum is a well-known medicinal mushroom. At present, numerous G. lucidum products have emerged in the form of dietary supplements in the United States due to its various benefits. However, the quality consistency of these products based on their label ingredients has seldom been evaluated due to the lack of a suitable toolkit. In this study, 19 batches of products of G. lucidum (Red Reishi, Reishi), herbal/mushroom supplements purchased in the United States, were evaluated based on their bioactive components including triterpenes and polysaccharides by using chromatographic methods and saccharide mapping. The results showed that the measured ingredients of only 5 tested samples (26.3%) were in accordance with their labels, which suggested the quality consistency of G. lucidum dietary supplements in the U.S. market was poor, which should be carefully investigated.

Quantitative analysis of flavonoids and phenolic acid in Coreopsis tinctoria Nutt. by capillary zone electrophoresis.

Capillary zone electrophoresis was developed for the simultaneous determination of five flavonoids and one phenolic acid, including taxifolin-7-O-glucoside, flavanomarein, quercetagetin-7-O-glucoside, okanin 4'-O-glucoside, okanin, and chlorogenic acid, in different parts and origins of Coreopsis tinctoria and its related species. Effects of acidity, running-buffer concentration, and modifier concentration were investigated to determine the optimum conditions for analyte determination. Analysis was performed within 18 min by using 50 mM borax buffer containing 15% acetonitrile as a modifier (pH 9.0) at 25 kV and 25°C. Hyperoside was used as internal standard for quantification. The method was accurate, simple, and repeatable, and was successfully applied to the analysis in 13 samples with satisfactory assay results. Results showed that C. tinctoria obviously differed from the related flower tea materials, "Hangju" and "Gongju". The parts (flowers, buds, seeds, stems, and leaves) of C. tinctoria also varied among one another. This study can serve as a foundation for the quality control and pharmacological evaluation of different parts of C. tinctoria and its related species.

Comparative metabolomics analysis for the compatibility and incompatibility of kansui and licorice with different ratios by UHPLC-QTOF/MS and multivariate data analysis.

Kansui, the root of Euphorbia kansui T.N. Liou ex T.P. Wang (Euphorbiaceae), is a well-known poisonous traditional Chinese medicine (TCM). However, many monographs of TCM indicated that it cannot be co-used with licorice, as kansui-licorice is a typical "eighteen incompatible" medicaments. Our previous studies have indicated that kansui was effective in treating malignant pleural effusion (MPE), and the efficacy could be weakened by the co-use of licorice, even causing serious toxicity at the given ratio. Nevertheless, the actual mechanisms of their dosage-toxicity-efficacy relationship need to be well clarified. The present study aimed to investigate the effect of individual and combined use of kansui and licorice on MPE rats, and explain the underlying mechanisms from a metabolomic perspective. Urine samples were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the interaction between kansui and licorice. Seven potential biomarkers contribute to the separation of model group and control group were tentatively identified. And selenoamino acid metabolism and nicotinate and nicotinamide metabolism with the impact-value 0.31 and 0.24, respectively, were filtered out as the most important metabolic pathways. Kansui and kansui-licorice at a ratio of 4:1 can treat MPE rats by adjusting abnormal metabolic pathways to the normal state, while it may have opposite result with kansui-licorice 1:4. The different influences to the two metabolic pathways may partially explain the dosage-toxicity-efficacy relationship of kansui-licorice with different ratios. The results could offer valuable insights into the compatibility property changes for the two herbs.

Identification of four cornua by ultra-performance liquid chromatography with time-of-flight mass spectrometry coupled with principal component analysis.

An ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin-digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least-squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.

Comparison of Immunomodulatory Effects of Fresh Garlic and Black Garlic Polysaccharides on RAW 264.7 Macrophages.

Garlic has a long history to be used for medicine and food purposes. Black garlic, the fermented product of fresh garlic, is considered with better biological activities, such as antioxidant activity, and is developed as an increasingly popular functional food. Polysaccharides are the major components of fresh and black garlic, and immunomodulatory activity is one major pharmacological effect of polysaccharides. Therefore, chemical characteristics and immunomodulatory effects of polysaccharides from fresh and black garlic are investigated and compared in vitro for the 1st time, in order to reveal their molecular and pharmacological differences. It is demonstrated that the molecular weights of polysaccharides from the 2 sources and molar ratios of monosaccharides after acid hydrolysis are greatly variant. The effects of polysaccharides from 2 sources on RAW 264.7 macrophages functions, including promotion of phagocytosis, release of NO, and expressions of several immune-related cytokines (including interleukin [IL]-6, IL-10, tumor necrosis factor alpha, and interferon gamma), were different from each other. The results indicated that fresh garlic polysaccharide exhibited stronger immunomodulatory activities than that of black garlic. Moreover, it is revealed that fructan might be the bioactive component in garlic and it is indicated that during the fermentation treatment, fructan constituents of garlic has degraded, and basically no immunomodulatory effect can be found in black garlic polysaccharides.