Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high‐throughput UHPLC–MS with a multivariate analysis approach

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in‐depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese medicine.     

Global detection and identification of components from crude and processed traditional Chinese medicine by liquid chromatography connected with hybrid ion trap and time‐of‐flight‐mass spectrometry

We herein present a chemical profiling method to efficiently process the information acquired by ultra fast liquid chromatography (UFLC)‐electrospray ionization source in combination with hybrid ion trap and high‐resolution time‐of‐flight mass spectrometry (UFLC‐(ESI)‐IT‐TOF/MS), facilitating the structural determination of serial components contained in crude or processed traditional Chinese medicine (TCM). Under the optimized UFLC and IT‐TOF‐MSⁿ conditions, over 39 compounds were separated and detected in crude or processed Fructus corni within 25 min. The components were identified by comparing the mass spectra and retention time with reference compounds, or tentatively assigned by elucidating low‐energy collision‐induced dissociation (CID) fragment ions and matching empirical molecular formula with that of the published compounds. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used to elucidate the processed mechanism of F. corni, which is regularly affected by the processing conditions. This study provides a novel approach and methodology to identify the complicated components from various complex mixtures such as crude TCM, processed TCM, and biological samples. It can be used as a valid analytical method for further understanding the processing mechanism of TCM, along with the intrinsic quality control of TCM and its processed product.     

Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry and chemometric analysis of licorice based on the simultaneous determination of saponins and flavonoids

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β‐glycyrrhizic acid and liquiritin. In addition, both 18β‐glycyrrhizic acid and licorice‐saponin G2 can be used for discrimination between crude and honey‐processed licorice. Furthermore, using 18β‐glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.     

Quality evaluation of raw and processed Crataegi Fructus by color measurement and fingerprint analysis

Crataegi Fructus and its processed products have been used as a traditional medicine for a long time, and numerous active components are responsible for their curative effects. However, a comprehensive and fast method for the quality control of its processed products is still lacking. In this study, two analytical methods based on color measurements and fingerprint analysis are established. In the color measurements, the color values of the peel and flesh of Crataegi Fructus were evaluated spectrophotometrically. Based on the results, a color reference range was established using percentiles, and the standard color difference value was established using the median color values. Then, the color values of Crataegi Fructus and its processed products were analyzed using Bayes linear discriminant analysis and mathematical functions were built in order to predict the degree of processing. Moreover, high‐performance liquid chromatography fingerprint analysis was performed on a Hibar C₁₈ column, and a high‐performance liquid chromatography fingerprint pattern was obtained, from which nine peaks were identified. Chemometric methods were successfully applied to differentiate raw and processed Crataegi Fructus.     

Studies on the Components of Crude and Processed Fructus Corni by ESI-MS

The components of crude and processed Fructus Corni were investigated by means of electrospray ionization-tandem mass spectrometry（ESI-MSn） technique in the negative ion mode. Compared with those of crude Fructus Corni, the chemical components of the processed Fructus Corni were changed both in quality and in quantity. From the ESI-MS spectra of the crude and processed Fructus Corni, six peaks were selected to establish the characte-ristic ESI-MS peaks. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used for the elucidation of the processed mechanism of Fructus Corni, which is regularly affected by the processing conditions. The present article provides both the chemistry evidence for the understanding of the processing procedure of Fructus Corni and the specific methodology for the research of the processing procedure and quality identification of traditional Chinese medicine.     

Comparative in vivo pharmacokinetics study of affeic acid,isoferulic acid and ferulic acid in crude and three different prepared Cimicifuga foetida L.

Our study investigated the differences in pharmacokinetics of three major components of crude Cimicifuga foetida L. and its fried product and honey- and liquor-prepared products. A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for determing caffeic acid, isoferulic acid and ferulic acid in rat plasma. The approach has good linearity, precision, accuracy, recovery and stability. Phenolic acid was rapidly absorbed. The times to peak concentration were shorter in the processed group than those for the crude product, with their values of <30 min. The peak concentration values of caffeic acid and isoferulic acid were higher in the crude group than in the processed groups (p < 0.05). Area under the curve values of the three phenolics in the crude group were significantly higher than those of the processed groups (p < 0.05).     

Quantitative Determination and Variation Trends of Six Alkaloids in Crude and Processed Corydalis Yanhusuo

A convenient, sensitive, and accurate analytical method using reversed-phase high-performance liquid chromatography coupled with ultraviolet-visible detection was investigated for the determination of 6 alkaloids (tetrahydrocoptisine, protopine, tetrahydropalmatine, coptisine, corydaline, and palmatine) in crude and processed samples of corydalis yanhusuo. The results revealed that the content of the alkaloids in crude corydalis yanhusuo samples varied from those of the processed samples. The concentrations of tetrahydrocoptisine, protopine, tetrahydropalmatine, coptisine, and corydaline were reduced after processing; however, the concentration of palmatine increased after processing. This method was established using a C₁₈ column (250 mm × 4.6 mm, 5 µm). The mobile phase, a mixture (19:81) of acetonitrile and a 0.6% solution of acetic acid (pH 3.5, adjusted using triethylamine), was used to elute the target compounds in the isocratic elution mode. The solvent flow rate was 1.0 mL/min, the column temperature was 35°C, and the detection wavelength was 280 nm. All of the calibration curves exhibited good linearity with R >0.9999. The inter- and intra-day precisions for all of the investigated components ranged from 0.10–2.64% RSD, and the recoveries were in the range of 97.3–102.5%. The validated method provides a new solution for analyzing the contents of crude and processed samples of corydalis yanhusuo.     

Qualitative and Quantitative Analysis of Alkaloids in Cortex Phellodendri by HPLC-ESI-MS/MS and HPLC-DAD

A combined method of high performance liquid chromatograph-elecrtrospray-ionization mass spectrometer(HPLC-ESI-MS/MS) coupled with a photodiode array detector(HPLC-DAD) and principal component analysis(PCA) was applied to the qualitative and quantitative analyses of alkaloids in Cortex Phellodendri(CP) samples, and to the differentiation of two species of CP, Cortex Phellodendri Chinensis(CPC) and Cortex Phellodendri Amurensis(CPA). Twenty-two peaks appeared in the HPLC-MS base peak chromatogram of CP detec...     

Study of organic acids in Schisandrae Chinensis Fructus after vinegar processing

The ripened fruit of Schisandrae Chinensis Fructus has unique medical properties in Chinese medicine. It is commonly used after vinegar steaming. Vinegar steaming changes the color of Schisandrae Chinensis Fructus from red to black and enhances its acidic and astringent properties. Lignans are the well‐investigated components in this herb. However, Schisandrae Chinensis Fructus is acidic in the theory of Chinese medicine, and whether vinegar processing changes its organic acid components remains largely unknown. In this study, the organic acids in this herb were derived by the method of methyl esterification, and further analyzed by gas chromatography with mass spectrometry. A total of 39 organic acid compounds were identified. Interestingly, Schisandrae Chinensis Fructus after vinegar processing showed a significant increase in the content of levulinic acid as compared to the unprocessed ones. Pharmacological experiments demonstrated that levulinic acid inhibited the contractility of isolated intestine and had an inhibitory effect on the excessive hyperfunction of small intestinal propulsion. Moreover, the extracts of vinegar‐processed Schisandrae Chinensis Fructus had a stronger inhibitory on the excessive hyperfunction of small intestinal propulsion than that of unprocessed ones. Taken together, this study offers novel insight into the effect of Schisandrae Chinensis Fructus after vinegar processing.     

Chemical composition analysis of Angelica sinensis (Oliv.) Diels and its four processed products by ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry combining with nontargeted metabolomics

Angelica sinensis (Oliv.) Diels. has been used for women to enrich the blood, prevent and treat blood deficiency syndrome in Traditional Chinese Medicine for thousands of years. Wine-processed Angelica sinensis, soil-processed Angelica sinensis, oil-processed Angelica sinensis, and charred-processed Angelica sinensis are the most significant four processed products used in Chinese clinic. However, there have been few studies aimed at comparing their chemical differences. Ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry combining with nontargeted metabolomics was applied to investigate the diversity of processed products of Angelica sinensis. A total of 74 compounds with the variable importance in the projection value more than 1.5 and P less than 0.05 in ANOVA were highlighted as the compounds that contribute most to the discrimination of Angelica sinensis and four processed products. The results showed the metabolic changes between Angelica sinensis and its four processed products, there were 19 metabolites, 3 metabolites, 6 metabolites, and 45 metabolites were tentatively assigned in soil-processed Angelica sinensis, wine-processed Angelica sinensis, oil-processed Angelica sinensis, and charred-processed Angelica sinensis, respectively. These results suggested that the proposed metabolomics approach was useful for the quality evaluation and control of processed products of Angelica sinensis.     

Quality assessment of raw and baked Aucklandia lappa Decne. by color measurement and fingerprint analysis

Aucklandia lappa Decne. has been used as a traditional Chinese herb for thousands of years in treating various kinds of disorders. According to the Chinese Pharmacopoeia, there are two kinds of processed products, raw and baked Aucklandia lappa Decne., which have different therapeutic effect in clinical application. In this study, based on color measurement and fingerprint analysis, the method to assess the quality of these two processed products was established. In color measurement, the reference ranges of color parameters (L^*, a^*, and b^*), standard color difference values, and mathematical prediction functions of these two processed products were obtain after the color was measured by a spectrophotometer. Meanwhile, high‐performance liquid chromatography fingerprints of these two processed products were established, where there were 12 peaks recognized as the common peaks in both processed products, in which two peaks were identified as costunolide and dehydrocostus lactone, and these two processed products were classified with chemometrics analysis subsequently. Furthermore, the correlation between color parameters and sample compositions was explored and the contents of costunolide and dehydrocostus lactone were determined simultaneously by high‐performance liquid chromatography. Consequently, an integral method including color measurement, high‐performance liquid chromatography fingerprint with chemometrics analysis, and quantitative determination was established.     

An integrated strategy of secondary metabolomics and glycomics to investigate multi-component variations in wine-processing of medicinal herbs and functional foods: A case study on Fructus Corni

Fructus Corni (FC), as a promising Chinese medicinal herb, has aroused considerable interest. Generally, FC needs to be processed according to the limited standard policy in China before clinical application, while the investigations on the specific processing methods (such as wine steaming or high-pressure wine steaming) are unclear. A comprehensive metabolomics strategy based on integrated non-targeted metabolomics and targeted glycomics in this paper was implemented to investigate the influences of the different processing technologies such as steaming, wine steaming, high-pressure steaming, high-pressure wine steaming, wine immersion, and wine stir-frying on FC, respectively. UHPLC-Q-TOF-MS/MS was employed for identifying and distinguishing the secondary metabolites. A total of 85 components were identified in all groups. The results of PCA score plots showed that the crude and processed samples had a complete separation, and wine steamed and high-pressure wine steamed samples could be a category, indicating that the two processed products had a similar quality. Multiple chromatography including HPLC (C₁₈)-PDA, HPLC (NH₂)-ELSD, and HPGPC-ELSD was used for determining the molecular weight distributions, the monosaccharide compositions of polysaccharides, and the contents of free monosaccharides and oligosaccharides. The results indicated that the content and composition of saccharides were different in crude and different processed FC. The polysaccharides were composed of fucose, arabinose, galactose, glucose, galacturonic acid, mannose and rhamnose, and the free monosaccharides were composed of fucose, arabinose, galactose, glucose, mannose, rhamnose and fructose in all FC samples. The PCA score plots of the glycomics indicated that the crude and high-pressure wine steamed FC could be a category, showing that the two groups had similar chemical compositions. Ultimately, the simulation processing experiments indicated that the transformation of morroniside, polysaccharides, oligosaccharides, fructose, and glucose to 5-HMF through the reactions of dehydration and deglycosylation was the potential mechanism of enhancing the effects by processing. Conclusionly, the saccharides should be investigated as thoroughly as the secondary metabolites, and the high-pressure wine steamed FC could be an alternative to wine steamed FC.     

New Approaches for the Synthesis of 1,3,4-Thiadiazole and 1,2,4-Triazole Derivatives with Antimicrobial Activity

The thiosemicarbazide derivatives 3a and 3b were cyclized in the presence of concentrated sulfuric acid to give the 5-cyanomethyl-1,3,4-thiadiazole derivatives 4a and 4b , respectively. The latter products were used for many heterocyclic transformations to form coumarin, 1,3,4-thiadiazolo[4,5-a]pyridine, and 5-thiophenylthiophene. In addition, compound 3b underwent cyclization in NaOH (2 N) solution to give the 1,2,4-triazole derivative 16 . The reactivity of the latter product towards some chemical reagents was studied. The antimicrobial activities of the newly synthesized products were measured and showed high activities.     

Cytotoxic Activities of Extracts and Compounds from <Emphasis Type="Italic">Viscum coloratum</Emphasis> and its Transformation Products by <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The bioassay-oriented fractionation of mistletoe crude extracts (MCEE) using 75% ethanol and culture products of mistletoe transformed by Rhodobacter sphaeroides, a photosynthetic bacterium (PSBT), revealed that the high cytotoxic activities were due to the petroleum ether extracts (PEs) and the acid-precipitated proteins from the aqueous extracts (AQs) of MCEE and PSBT. The isolated triterpenes may account for the activities of the PEs of MCEE and PSBT, respectively. Extraction of MCEE using petroleum ether led to the isolation of 3-epi-betulinic acid (1), betulonic acid (2), oleanolic acid (3), and β-amyrin acetate (4), while petroleum ether extraction of PSBT led to the isolation of 1,3,4,betulinic acid (5), erythrodiol (6), and (3β)-olean-12-ene-3,23-diol (7). The PE of PSBT exerted higher cytotoxicity than the PE of MCEE, which was due to the different triterpene contents of these two extracts. The cytotoxic activities of all compounds were tested, and the results revealed that compounds 1, 2, 3, 5, 6, and 7 contributed significantly to the cytotoxicities of both PEs. The AQ of the PSBT exerted almost the same cytotoxic activity and lower toxicity compared to the AQ of the MCEE. These findings indicate that mistletoe products biotransformed by R. sphaeroides could be used to treat cancers, since they have lower toxicities and higher antitumor activities compared to standard treatments.     

Changes in Triterpenes in Alismatis rhizoma after Processing Based on Targeted Metabolomics Using UHPLC-QTOF-MS/MS

Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People′s Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications.     

Comparison of the active components of Aster tataricus from different regions and related processed products by ultra‐high performance liquid chromatography with tandem mass spectrometry

We investigated crude Aster tataricus, vinegar‐processed Aster tataricus, honey‐processed Aster tataricus, and steamed Aster tataricus as a case study and developed a comprehensive strategy integrating quantitative analysis and chemical pattern recognition methods for the evaluation and differentiation of Aster tataricus from different regions, as well as related processed products. In the study, 15 batches of raw Aster tataricus collected from seven provinces were analyzed. A sensitive and rapid ultra‐high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of 15 compounds was established to evaluate the quality of raw and processed Aster tataricus. Furthermore, multivariate statistical techniques were applied to compare the differences among Aster tataricus samples. As a result, the herbs collected from seven provinces were divided into two categories, and chlorogenic acid was the most important component distinguishing between the regions. Moreover, all of the raw and processed samples were classified by partial least squares discriminant analysis based on the 15 analyzed compounds. Results showed that raw Aster tataricus, vinegar‐processed Aster tataricus, honey‐processed Aster tataricus, and steamed Aster tataricus were clustered in four different areas. Shionone, chlorogenic acid and kaempferol were the significant constituents differentiating the raw and differently processed Aster tataricus samples.     

Analysis of dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and processed seeds by high‐performance liquid chromatography coupled with diode array detection and mass spectrometry

The ripened seeds of Strychnos nux‐vomica L. have been extensively used as herbal medicines in Asian countries. Dihydroindole‐type alkaloids are not only the active constituents but also the toxicants in Strychnos. However, the simultaneous determination of these alkaloids in both crude and processed Semen Strychni is still lacking. The present study represents the first quantitation and relative quantitation assay of 12 dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and sand‐processed seeds using high‐performance liquid chromatography coupled with diode array detection and mass spectrometry. The relative concentration of ten alkaloids was calculated by semi‐quantification using the internal standard and their amounts in unprocessed and detoxified Semen Strychni were compared. We report here for the first time the significant increase of the two alkaloids, 19‐N‐methyl‐strychnine, and 2,3‐dimethoxy‐19‐N‐methyl‐strychnine, during the processing of Semen Strychni. Our study provides new insight into the true complexity of seed processing procedure and valuable information for assessing the efficacy and safety for clinical applications of Semen Strychni‐containing drugs.     

A biochemometrics strategy combining quantitative determination,bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar‐processed Corydalis turtschaninovii

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar‐processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C₁₈ column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra‐day and inter‐day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar‐processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar‐processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar‐processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.     

A liquid chromatography–tandem mass spectrometry approach for study the tissue distributions of five components of crude and salt‐processed Radix Achyranthes in rats

This study developed a robust and reliable approach using liquid chromatography– tandem mass spectrometry for the simultaneous determination of five saponins in rat tissues: β‐ecdysterone, chikusetsusaponin IV, ginsenoside Ro, 25S‐inokosterone and chikusetsusaponin IVa. This is the first report on a comparative tissue distribution study of crude and salt‐processed Radix Achyranthes in rats. After one‐step protein precipitation by acetonitrile, the tissue samples were sent to LC–MS/MS for multiple reaction monitoring. The retention times of the five saponins and internal standard were 1.77, 3.14, 3.01, 1.83, 3.26 and 4.77 min. The standard curves showed good linear regression (r² > 0.9991) in the range of 10.3–1562.5 ng/mL. The intra‐ and inter‐day accuracy and precision were within 15% of the nominal concentration. The recoveries of the five saponins were 92.0–99.9%. Finally, this approach was successfully applied to tissue distribution analysis of the five saponins after oral administration of crude and salt‐processed Radix Achyranthes in rats. The largest concentration of the five saponins was observed in kidney after salt‐processing, which indicated that processing could enhance the bioavailability.     

Synthesis of New Plant Growth Regulators,Isomeric 1-(Dimethyloctyl)- and 1-Alkylpiperidinium and -morpholinium Halides by Telomerization of Isoprene with Secondary Amines

The known plant growth regulator, Al'den [1-(3,7-dimethyloctyl)-1-allylpiperidinium bromide], and also 1-(3,7-dimethyloctyl)-1-methylpiperidinium iodide were synthesized by treatment with allyl bromide and methyl iodide, respectively, of exhaustively hydrogenated 1-nerylpiperidine which was obtained by anionic telomerization of isoprene with piperidine. Various quaternary ammonium salts having a terpene sub- stituent with unnatural dimethyloctane skeleton, which effectively stimulate florification of Aster Chinensis L., were prepared by the action of alkyl halides on telomerization products derived from isoprene and piperidine or morpholine in the presence of palladium complexes.