The difference between blood‐associated and water‐associated herbs of Danggui‐Shaoyao San in theory of TCM,based on serum pharmacochemistry

Danggui‐Shaoyao San (DSS) is a famous Chinese formula for activating blood circulation and promoting urination. This study was to investigate the difference of material basis between a blood‐associated herbs group and a water‐associated herbs group. According to the theory of traditional Chinese medicine, the formula can be divided into a blood‐associated herbs group (Angelica sinensis, Paeonia lactiflora and Ligusticum chuanxiong) and a water‐associated herbs group (Atractylodes macrocephala, Alisma orientale and Poria cocos). The HPLC fingerprint of the formula was established for quality control. Serum samples from rats, orally administrated DSS, and the decomposed recipes of DSS, were analyzed by HPLC‐DAD and the transitional blood components of DSS were identified. Twenty‐one common peaks were identified in the fingerprint of DSS. Contents of paeoniflorin, albiflorin, ferulic acid and alisol B 23‐acetate in co‐decoction were significantly higher than those in individual decoction. Eleven peaks belonged to the blood‐associated herbs group (four metabolites and seven prototype components; paeoniflorin and ferulic acid appeared in prototype components), whereas six peaks belonged to the water‐associated herbs group (three metabolites and three prototype components). It was concluded that the serum pharmacochemistry is a meaningful approach for clarifying the difference between blood‐associated and water‐associated herbs in chemical composition. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of interactions between multiple components in Socheongryong‐tang using a plant profiling approach

Traditional herbal medicine consists of multiple components. There are interactions among the components, which affect both potency and toxicity. The preparation of herbal medicines can be a cause of interactions between multicomponents in herbs. To demonstrate the differences in multiherb interactions based on the preparation methods, the changes in the active components in the different preparations of Socheongryong‐tang (SCRT) were evaluated using metabolomics profiling. We performed multicomponent profiling of the decoction of SCRT (SCRTD) and individual herb mixture (SCRTM) using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UPLC–QTOF–MS). Active compounds from SCRTD and SCRTM were identified using multivariate analysis, and the activities between the two groups were compared. We also evaluated the anti‐inflammatory effect of SCRT through investigating the protein expression of iNOS and COX‐2 in lipopolysaccharide‐induced macrophage RAW 264.7 cells in both groups. From the multivariate analysis, 53 active compounds that have different intensities between SCRTD and SCRTM were identified. The intensities of those components, such as ephedrines, glycyrrhizic acid, 6‐gingerol and (2E,4E,8Z,10E)‐N‐isobutyl‐2,4,8,10‐dodecatetraenamide, which is newly identified in Asiasarum heterotropoides, were mostly higher in SCRTD than in SCRTM, which was related to the anti‐inflammatory effect. From the iNOS inhibition test, it was found that SCRTD had a stronger anti‐inflammatory effect than SCRTM. It was demonstrated that multicomponent interactions can be changed by the preparation method, and finally the anti‐inflammatory effect in SCRT can be affected.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.     

Chemical interaction between Lilium brownii and Rhizoma Anemarrhenae,the herbal constituents of Baihe Zhimu decoction,by liquid chromatography coupled to hybrid triple quadrupole linear ion trap mass spectrometer

During the course of decoction, the components of herbal formula interact with each other, such that chemical extraction characteristics are altered. The crude drugs, Lilium brownii (Baihe) and Rhizoma Anemarrhenae (Zhimu), are the herbal constituents of Baihe Zhimu decoction, a traditional herbal formula. To investigate the chemical interaction between Baihe and Zhimu when decocting together, eight marker components in Baihe Zhimu decoction were simultaneously characterized and quantified in one run by a hybrid triple quadrupole linear ion trap mass spectrometer in the multiple reactions monitoring–information dependent acquisition–enhanced product ion mode. The results showed that Zhimu significantly suppressed the extraction of phenolic glycosides (the components from Baihe) when co‐decocting, and Baihe clearly suppressed the extraction of xanthones and steroidal saponins (the components from Zhimu). Overall, the presently developed method would be a preferred candidate for the investigation of the chemical interaction between herbal medicines.     

Characterization of chemical constituents in Zhi–Zi–Da–Huang decoction by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to separate and identify the chemical constituents of Zhi–Zi–Da–Huang decoction, a classic traditional Chinese medicine formula. The chromatographic separation was achieved on a Shim‐pack XR‐ODS C₁₈ column (75  × 3.0 mm, 2.2 μm) using a gradient elution program. The detection was performed on a Waters Xevo G2 Q‐TOF mass spectrometer equipped with electrospray ionization source in both positive and negative modes. With the optimized conditions, a total of 82 compounds were identified or tentatively characterized. Of the 82 compounds, 21 compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literature. In addition, 31 compounds were identified from Gardenia jasminoides Ellis, ten compounds were identified from Rheum palmatum L., 33 compounds were identified from Citrus aurantium L., and eight compounds were identified from Sojae Semen Praeparatum. Results indicated that iridoids, anthraquinones, flavonoids, isoflavonoids, coumarins, glycosides of crocetin, monoterpenoids, and organic acids were major constituents in Zhi–Zi–Da–Huang decoction. It is concluded that the developed ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Zhi–Zi–Da–Huang decoction, and the analysis provides a helpful chemical basis for further research on Zhi–Zi–Da–Huang decoction.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

Effects of PAR and UV‐B Radiation on Herbal Yield,Bioactive Compounds and Their Antioxidant Capacity of Some Medicinal Plants Under Controlled Environmental Conditions

Photosynthetically active radiation (PAR) and Ultraviolet B (UV‐B) radiation are among the main environmental factors acting on herbal yield and biosynthesis of bioactive compounds in medicinal plants. The objective of this study was to evaluate the influence of biologically effective UV‐B light (280–315 nm) and PAR (400–700 nm) on herbal yield, content and composition, as well as antioxidant capacity of essential oils and polyphenols of lemon catmint (Nepeta cataria L. f. citriodora), lemon balm (Melissa officinalis L.) and sage (Salvia officinalis L.) under controlled greenhouse cultivation. Intensive UV‐B radiation (2.5 kJ m^?2 d^?1) influenced positively the herbal yield. The essential oil content and composition of studied herbs were mainly affected by PAR and UV‐B radiation. In general, additional low‐dose UV‐B radiation (1 kJ m^?2d^?1) was most effective for biosynthesis of polyphenols in herbs. Analysis of major polyphenolic compounds provided differences in sensitivity of main polyphenols to PAR and UV‐B radiation. Essential oils and polyphenol‐rich extracts of radiated herbs showed essential differences in antioxidant capacity by the ABTS system. Information from this study can be useful for herbal biomass and secondary metabolite production with superior quality under controlled environment conditions.     

Rapid identification of the quality decoction pieces by partial least squares ‐based pattern recognition: grade classification of the decoction pieces of Saposhnikovia divaricata

Herbal medicines are commonly used in many countries after they undergo processing. Quality decoction pieces are a guarantee of the efficacy and safety of the herbal medical products. Here, a strategy based on chemical analysis combined with chemometric techniques was proposed for the classification and prediction of the different grades of the decoction pieces. Considering the necessity for a shared and simple method for the grade classification for the public, in this paper, the characterization of the chemical constituents was determined by utilizing high‐performance liquid chromatography (HPLC)/diode array detection. HPLC was first established for the characterization of the chemical constituents of the different grade decoction pieces. Furthermore, a simultaneous quantification of several of the marker compounds in these decoction pieces was obtained. Finally, a partial least squares‐based pattern recognition method was utilized to obtain a predictive model for the grade classification of the decoction pieces. Saposhnikovia divaricata (Turcz.) Schischk was used as a case study. The partial least squares ‐based pattern recognition for the grade classification of the decoction pieces of S. divaricata demonstrated good sensitivity, specificity and prediction performance, which may efficiently validate the identification results of appearance assessment. The proposed strategy is expected to provide a new insight for the grade classification and quality control of the decoction pieces. Copyright © 2016 John Wiley & Sons, Ltd.     

Chemical profiling approach to evaluate the influence of traditional and simplified decoction methods on the holistic quality of Da‐Huang‐Xiao‐Shi decoction using high‐performance liquid chromatography coupled with diode‐array detection and time‐of‐flight mass spectrometry

Da‐Huang‐Xiao‐Shi decoction, consisting of Rheum officinale Baill, Mirabilitum, Phellodendron amurense Rupr. and Gardenia jasminoides Ellis, is a traditional Chinese medicine used for the treatment of jaundice. As described in “Jin Kui Yao Lue”, a traditional multistep decoction of Da‐Huang‐Xiao‐Shi decoction was required while simplified one‐step decoction was used in recent repsorts. To investigate the chemical difference between the decoctions obtained by the traditional and simplified preparations, a sensitive and reliable approach of high‐performance liquid chromatography coupled with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry was established. As a result, a total of 105 compounds were detected and identified. Analysis of the chromatogram profiles of the two decoctions showed that many compounds in the decoction of simplified preparation had changed obviously compared with those in traditional preparation. The changes of constituents would be bound to cause the differences in the therapeutic effects of the two decoctions. The present study demonstrated that certain preparation methods significantly affect the holistic quality of traditional Chinese medicines and the use of a suitable preparation method is crucial for these medicines to produce special clinical curative effect. This research results elucidated the scientific basis of traditional preparation methods in Chinese medicines.     

Identification of bioactive compounds in Shaoyao‐Gancao decoction using β2‐adrenoceptor affinity chromatography

Shaoyao‐Gancao decoction, a Chinese herbal formula, is composed of Paeoniae Radix alba and Glycyrrhiza Radix et rhizoma . It has been widely used to treat muscle spasms and asthma. However, little is known about the bioactive components of Shaoyao‐Gancao decoction. In the present study, the bioactive compounds in water‐extract of Shaoyao‐Gancao decoction were separated by the immobilized β₂‐adrenoceptor affinity column and identified using quadrupole time‐of‐flight mass spectrometry. The affinity constants of the separated compounds that bind to β₂‐adrenoceptor were determined by frontal analysis. Compound bioactivity was tested in a rat tracheal smooth muscle relaxation assay. We identified the bioactive compounds in the water extract of Shaoyao‐Gancao decoction that bound to the β₂‐adrenoceptor as paeoniflorin and liquiritin. Paeoniflorin and liquiritin had only one binding site on the immobilized β₂‐adrenoceptor, and the affinity constants were (2.16 ± 0.10) × 10⁴ M⁻¹ and (2.95 ± 0.15) × 10⁴ M⁻¹, respectively. Both compounds induced a concentration‐dependent relaxation of tracheal smooth muscle following K⁺‐stimulated contraction, and the relaxation effects were abrogated by the β₂‐adrenoceptor antagonist, ICI 118551. Therefore, paeoniflorin and liquiritin are bioactive compounds in Shaoyao‐Gancao decoction and the β₂‐adrenoceptor affinity chromatography is a useful tool for identifying potential β₂‐adrenoceptor ligands in natural products used in traditional Chinese medicine.     

Combination of high‐resolution accurate mass spectrometry and network pharmacology provides a new method for the chemical constituents’ study and target prediction in vivo of Garcinia multiflora

Garcinia multiflora is a kind of evergreen tree which is widely distributed in the south of China. However, few researches focused on the constituents in different parts of G. multiflora as well as their potential targets and pathways in vivo. To clarify the chemical constituents of G. multiflora rapidly and predict the potential targets as well as pathways in vivo that this plant may have effects on, a feasible and accurate strategy was developed to identify the chemical constituents in fruits, leaves, and branches of G. multiflora by ultra‐high performance liquid chromatography with Q‐Exactive hybrid quadrupole‐orbitrap high‐resolution accurate mass spectrometry. Network pharmacology was then employed and a “compounds‐targets‐diseases” network was established. Sixty‐one compounds including polycyclic polyprenylated acylphloroglucinols, xanthones, and flavonoids were finally identified in different parts of G. multiflora, and the contents of seven constituents were quantified, respectively. On the basis of the network pharmacology analysis results, compounds in this plant were speculated to have potential pharmacodynamic effect on cancer, inflammatory, respiratory diseases, cardiovascular diseases, and metabolic diseases. This research will provide a new method for the advanced study on the pharmacodynamic materials basis of G. multiflora, and offer valuable evidences for medicinal purpose of this plant.     

Comparative analysis of volatile constituents between recipe jingfangsan and its single herbs by GC‐MS combined with alternative moving window factor analysis method

Comparative analysis of volatile constituents between recipe jingfangsan and its single herbs was performed by GC‐MS combined with alternative moving window factor analysis (AMWFA), a new chemometric resolution method. Identification of the compounds was also assisted by comparison of temperature‐programmed retention indices (PTRIs) on the OV‐1 column with authentic samples. In total, 36, 29, and 42 volatile components in essential oil of Herba schizonepetae (HS), Radix saposhnikoviae (RS), and the recipe were respectively determined qualitatively and quantitatively, accounting for 81.80, 82.62 and 85.98% total contents of volatile oil of HS, RS, and the recipe respectively. Analysis by the method of AMWFA indicates that there are 22 common volatile constituents between the recipe and single herbal medicine HS, and 14 common volatile constituents between the recipe and single herb medicine RS. The experimental results also show that the volatile components of the recipe in number are almost addition of that of two single herbal medicines HS and RS, and are mainly from the single herbal medicine HS.     

Strategy for rapid screening of antioxidant and anti‐inflammatory active ingredients in Gynura procumbens (Lour.) Merr. based on UHPLC–Q‐TOF–MS/MS and characteristic ion filtration

Gynura procumbens (Lour.) Merr. is traditionally used as a raw material for making dumplings or steamed stuffed buns, and its fresh leaves are boiled with water for tea. Herein, we established an ultra‐high–performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC–Q‐TOF–MS/MS) combined with characteristic ion filtration (CIF) strategy to rapidly screen active ingredients with antioxidant and anti‐inflammatory properties in G. procumbens. This strategy involved screening the active part of G. procumbens using antioxidation and anti‐inflammatory activity assays; discovering the active compounds by speculating on the active site's chemical composition by UHPLC–Q‐TOF–MS/MS plus CIF; and verifying the active compounds' activities. The ethyl acetate extract (EEAF) of G. procumbens was the major active site. Eighty‐one compounds were identified from the EEAF using UHPLC–Q‐TOF–MS/MS plus CIF. Furthermore, polyphenols such as cynarine, isochlorogenic acids A and isochlorogenic acids C have excellent antioxidizing and anti‐inflammatory activities. This study provides a practical strategy for rapid in vitro screening of the antioxidizing and anti‐inflammatory activities of traditional vegetables and herbs and identification of active ingredients.     

Chemical constituents and quality control of two Dracocephalum species based on high‐performance liquid chromatographic fingerprints coupled with tandem mass spectrometry and chemometrics

Two similar Dracocephalum species, namely, Dracocephalum tanguticum Maxim and Dracocephalum moldavica L, are commonly used as ethnic medicines in China. Here we describe a strategy of combining high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry, as well as fingerprints and chemometrics for characterization and discrimination of chemical constituents on the two herbs. A total of 49 compounds including 33 flavonoids, 5 phenylethanol glycosides, 1 coumarin glycoside, 8 organic acids, and 2 other types of compounds were unambiguously or tentatively identified from the two Dracocephalum species. Among the compounds identified, 26 were characterized for the first time and 4 compounds, rosmarinic acid ( 7 ), salvianolic acid B ( 10 ), luteoloside ( 22 ), diosmetin‐7‐O‐glucoside ( 28 ), were inferred as common constituents for the two herbs. Flavonoids featured in these two Dracocephalum species while their types presented significant differences. Acacetin ( 45 ) and acacetin glycosides (acatetin‐7‐O‐glucuronide ( 30 ), acacetin‐7‐O‐(6”‐O‐malonyl) glucoside ( 33 ), buddleoside ( 34 ), tilianin ( 35 ), and agastachoside ( 42 )) were detected only in D. moldavica, which can be used to discriminate two herbal medicines. In addition, six characteristic constitutes in D. tanguticum were simultaneously quantified. Moreover, the induced chemometrics methods including similarity analysis and hierarchical clustering analysis were successfully applied for origin discrimination and quality evaluation of D. tanguticum and D. moldavica.     

Identification of the absorptive constituents and their metabolites in vivo of Puerariae Lobatae Radix decoction orally administered in WZS‐miniature pigs by HPLC‐ESI‐Q‐TOFMS

In this study, the technique of high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (HPLC‐ESI‐Q‐TOFMS) was used to analyze and identify the absorptive constituents and their metabolites in drug‐containing urine of Wuzhishan (WZS)‐miniature pigs administered with Puerariae Lobatae Radix (PLR) decoction. With the accurate mass measurements (<5 ppm) and effective MS² fragment ions, 96 compounds, including eight original constituents and 88 metabolites, were identified from the drug‐containing urine. Among these, 64 metabolites were new ones and their structures can be categorized into five types: isoflavones, puerols, O‐desmethylangolensins, equols and isoflavanones. In particular, puerol‐type constituents in PLR were first proved to be absorptive in vivo. Meanwhile, the metabolic pathways of PLR in vivo were investigated. On the basis of relative content of the identified compounds, 13 major metabolites accounting for approximately 50% of the contents, as well as their corresponding 12 prototype compounds, were determined as the major original absorptive constituents and metabolites of PLR in vivo. The HPLC‐ESI‐Q‐TOFMS technique proved to be powerful for characterizing the chemical constituents from the complicated traditional Chinese medicine matrices in this research. Copyright © 2013 John Wiley & Sons, Ltd.     

Comprehensive identification of potential antioxidant components in the aerial parts of Polygonum chinense L. var. hispidum using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

The aerial parts of Polygonum chinense L. var. hispidum are one of the key herbs in Cantonese herbal tea, which is quite a common local beverage in LingNan area of China. Previous investigation has found that this herb possesses antioxidant activity and the ethyl acetate fraction of its ethanol extract shows the strongest antioxidant activity. However, little is known about its antioxidant chemical constituents. The aim of this research was to investigate the active constituents of this plant by identifying and characterizing the chemical profile in ethyl acetate fraction using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry, which can provide characteristic ultraviolet absorption, accurate molecular weight, and diagnostic tandem mass spectrometry fragment ions. As a result, 85 compounds were identified including 22 flavonoids, 12 ellagic acids, 34 ellagitannins, 16 phenolic acids, and one phenolic amide. All the phenolic compounds identified in this work, especially ethyl gallate, geraniin, chebulagic acid, and quercitrin with the higher peak areas in the ultra high performance liquid chromatography with mass spectrometry chemical profile of this plant, could be the bioactive principles responsible for the antioxidant activity. These findings in the present study could benefit further studies involving the functions and chemicals of this plant, and provide scientific evidence for usage of Cantonese herbal tea.     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.