Study on changes in pigment composition during the blooming period of safflower based on plant metabolomics and semi-quantitative analysis.

Red and yellow pigments are the major ingredients of safflower, often used to color food and cosmetics. Carthamin was the main component of red pigment and hydroxysafflor yellow A and anhydrosafflower yellow B were representative components of yellow pigment. Plant metabolomics and semi-quantitative analysis were used to analyze the changes of pigment composition during the blooming period, especially these characteristic components. Carthamin, hydroxysafflor yellow A, anhydrosafflower yellow B, and other components were screened out as differential metabolites based on plant metabolomics. Then semi-quantitative analysis was used to quantify these three representative components of pigments. Experimental results showed that the content of pigments has dynamic changes along with flowering, in the early blooming period, yellow pigment accumulated much and red pigment was low in content. In the middle period, the accumulation rate of the yellow pigment slowed down and content was stabilized. In the next step, the content of yellow pigments gradually decreased, and the content of red pigments gradually increased. Later, the level of yellow pigment decreased significantly, and the accumulation rate of red pigment increased significantly. Last, the appearance color of safflower was red, with yellow parts barely visible, and accumulation of red pigment was the highest and of the yellow pigment was the lowest in content.

Analysis and evaluation of nucleosides, nucleobases, and amino acids in safflower from different regions based on ultra high performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry.

Safflower has both medicinal and edible values but research on its nutrient composition is still lacking. This study was established for the quantitative determination of 28 nucleosides, nucleobases, and amino acids based on the ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry. Analysis of 30 batches of safflower from different producing areas indicated that the contents of l-proline, l-asparagine, l(+)-arginine, l-serine, l-histidine, uracil, guanosine, and uridine was high in safflower. Principle component analysis and cluster analysis found that samples from different regions could be distinguished well, and samples from the same area could be clustered into one class, different geographical environments may cause the differences of nucleosides, nucleobases, and amino acids in safflower. The analysis of principal component analysis, cluster analysis, and counter propagation artificial neural network show similar results. Then the content of nucleosides, nucleobases, and essential amino acids were compared, and found that the content in safflower from Gansu was higher than those from other regions, and there was a little difference between the samples from Xinjiang, Sichuan, and Yunnan. This research revealed the composition of nucleosides, nucleobases, and amino acids in safflower, and provided a theoretical basis for utilization of safflower.

Gut microbiota modulation with traditional Chinese medicine: A system biology-driven approach.

With the development of system biology, traditional Chinese medicine (TCM) is drawing more and more attention nowadays. However, there are still many enigmas behind this ancient medical system because of the arcane theory and complex mechanism of actions. In recent decades, advancements in genome sequencing technologies, bioinformatics and culturomics have led to the groundbreaking characterization of the gut microbiota, a 'forgotten organ', and its role in host health and disease. Notably, gut microbiota has been emerging as a new avenue to understanding TCM. In this review, we will focus on the structure, composition, functionality and metabolites of gut microbiota affected by TCM so as to conversely understand its theory and mechanisms. We will also discuss the potential areas of gut microbiota for exploring Chinese material medica waste, Chinese marine material medica, add-on therapy and personalized precise medication of TCM. The review will conclude with future perspectives and challenges of gut microbiota in TCM intervention.

The Comprehensive Evaluation of Safflowers in Different Producing Areas by Combined Analysis of Color, Chemical Compounds, and Biological Activity.

In the present study, a new strategy including the combination of external appearance, chemical detection, and biological analysis was proposed for the comprehensive evaluation of safflowers in different producing areas. Firstly, 40 batches of safflower samples were classified into class I and II based on color measurements and K-means clustering analysis. Secondly, a rapid and sensitive analytical method was developed for simultaneous quantification of 16 chromaticity-related characteristic components (including characteristic components hydroxysafflor yellow A, anhydrosafflor yellow B, safflomin C, and another 13 flavonoid glycosides) in safflowers by ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (UPLC-QTRAP®/MS2). The results of the quantification indicate that hydroxysafflor yellow A, anhydrosafflor yellow B, kaempferol, quercetin, and safflomin C had significant differences between the two types of safflower, and class I of safflower had a higher content of hydroxysafflor yellow A, anhydrosafflor yellow B, and safflomin C as the main anti-thrombotic components in safflower. Thirdly, chemometrics methods were employed to illustrate the relationship in multivariate data of color measurements and chromaticity-related characteristic components. As a result, kaempferol-3-O-rutinoside and 6-hydroxykaempferol-3-O-ß-d-glucoside were strongly associated with the color indicators. Finally, anti-thrombotic analysis was used to evaluate activity and verify the suitability of the classification basis of safflower based on the color measurements. It was shown that brighter, redder, yellower, more orange-yellow, and more vivid safflowers divided into class I had a higher content of characteristic components and better anti-thrombotic activity. In summary, the presented strategy has potential for quality evaluation of other flower medicinal materials.

A Ferulic Acid Derivative FXS-3 Inhibits Proliferation and Metastasis of Human Lung Cancer A549 Cells via Positive JNK Signaling Pathway and Negative ERK/p38, AKT/mTOR and MEK/ERK Signaling Pathways.

Lung cancer is one of the most common malignancies and is an increasing cause of cancer-related deaths. In our previous study, a series of ferulic acid (FA) derivatives were designed and synthesized; they exhibited positive anti-cancer activities, especially for a compound labelled FXS-3. In this study, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, wherein it revealed the inhibitory effect of FXS-3 on the proliferation and metastasis of human lung cancer A549 cells. The further flow cytometry assay showed that FXS-3 induced apoptosis of A549 cells induced cell cycle arrest at the G0/G1 phase. The trans-well migration and Matrigel invasion assays revealed that FXS-3 inhibited the migration and invasion of A549 cells. By the western blotting analysis, FXS-3 increased the expression of B-cell lymphoma-2 (Bcl-2) associated X protein (Bax)/Bcl-2 ratio, inhibited matrix metalloproteinase (MMP)-2 and MMP-9, and regulated the extracellular signal-regulated kinase (ERK)/p38, c-Jun N-terminal kinase (JNK), protein kinase B (AKT)/mechanistic target of rapamycin (mTOR), as well as mitogen-activated protein kinase (MEK)/ERK signaling pathways. The subsequent A549 xenograft-bearing mouse model and tail vein injection of A549 cells induced pulmonary tumor metastasis model showed that FXS-3 significantly restrained the tumor growth and metastasis. In conclusion, FXS-3 might inhibit proliferation and metastasis of human lung cancer A549 cells by positively regulating JNK signaling pathway and negativly regulating ERK/p38, AKT/mTOR, and MEK/ERK signaling pathways, which provides important scientific basis for the development of anti-cancer drugs about FA derivatives.

Comparative analysis of main bio-active components in the herb pair Danshen-Honghua and its single herbs by ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry.

A sensitive, reliable, and powerful ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method was developed for simultaneous quantification of the 15 main bio-active components including phenolic acids and flavonoids within 13 min for the first time. The proposed method was first reported and validated by good linearity (r2  > 0.9975), limit of detection (1.12-7.01 ng/mL), limit of quantification (3.73-23.37 ng/mL), intra- and inter-day precisions (RSD ≤ 1.92%, RSD ≤ 2.45%), stability (RSD ≤ 5.63%), repeatability (RSD ≤ 4.34%), recovery (96.84-102.12%), and matrix effects (0.92-1.02). The established analytical methodology was successfully applied to comparative analysis of main bio-active components in the herb pair Danshen-Honghua and its single herbs. Compared to the single herb, the content of most flavonoid glycosides was remarkably increased in their herb pair, and main phenolic acids were decreased, conversely. The content changes of the main components in the herb pair supported the synergistic effects on promoting blood circulation and removing blood stasis. The results provide a scientific basis and reference for the quality control of Danshen-Honghua herb pair and the drug interactions based on variation of bio-active components in herb pairs.

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.

[Comparative study on promoting blood effects of Danshen-Honghua herb pair with different preparations based on chemometrics and multi-attribute comprehensive index methods].

To evaluate the promoting blood circulation and removing blood stasis effects of Danshen-Honghua(DH) herb pair with different preparations (alcohol, 50% alcohol and water) on blood rheology and coagulation functions in acute blood stasis rats, and optimize the best preparation method of DH based on principal component analysis(PCA), hierarchical cluster heatmap analysis and multi-attribute comprehensive index methods. Ice water bath and subcutaneous injection of adrenaline were both used to establish the acute blood stasis rat model. Then the blood stasis rats were administrated intragastrically with DH (alcohol, 50% alcohol and water) extracts. The whole blood viscosity(WBV), plasma viscosity(PV), erythrocyte sedimentation rate(ESR) and haematocrit(HCT) were tested to observe the effects of DH herb pair with different preparations and doses on hemorheology of blood stasis rats; the activated partial thromboplastin time(APTT), thrombin time(TT), prothrombin time(PT), and plasma fibrinogen(FIB) were tested to observe the effects of DH herb pair with different preparations on blood coagulation function and platelet aggregation of blood stasis rats. Then PCA, hierarchical cluster heatmap analysis and multi-attribute comprehensive index methods were all used to comprehensively evaluate the total promoting blood circulation and removing blood stasis effects of DH herb pair with different preparations. The hemorheological indexes and coagulation parameters of model group had significant differences with normal blank group. As compared with the model group, the DH herb pair with different preparations at low, middle and high doses could improve the blood hemorheology indexes and coagulation parameters in acute blood stasis rats with dose-effect relation. Based on the PCA, hierarchical cluster heatmap analysis and multi-attribute comprehensive index methods, the high dose group of 50% alcohol extract had the best effect of promoting blood circulation and removing blood stasis. Under the same dose but different preparations, 50% alcohol DH could obviously improve the hemorheology and blood coagulation function in acute blood stasis rats. These results suggested that DH herb pair with different preparations could obviously ameliorate the abnormality of hemorheology and blood coagulation function in acute blood stasis rats, and the optimized preparation of DH herb pair on promoting blood effects was 50% alcohol extract, providing scientific basis for more effective application of the DH herb pair in modern clinic medicine.

Comparative Analysis of the Effects of Hydroxysafflor Yellow A and Anhydrosafflor Yellow B in Safflower Series of Herb Pairs Using Prep-HPLC and a Selective Knock-Out Approach.

The flower of Carthamus tinctorius L. (Carthami Flos, safflower), important in traditional Chinese medicine (TCM), is known for treating blood stasis, coronary heart disease, hypertension, and cerebrovascular disease in clinical and experimental studies. It is widely accepted that hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) are the major bioactive components of many formulae comprised of safflower. In this study, selective knock-out of target components such as HSYA and ASYB by using preparative high performance liquid chromatography (prep-HPLC) followed by antiplatelet and anticoagulation activities evaluation was used to investigate the roles of bioactive ingredients in safflower series of herb pairs. The results showed that both HSYA and ASYB not only played a direct role in activating blood circulation, but also indirectly made a contribution to the total bioactivity of safflower series of herb pairs. The degree of contribution of HSYA in the safflower and its series herb pairs was as follows: Carthami Flos-Ginseng Radix et Rhizoma Rubra (CF-GR) > Carthami Flos-Sappan Lignum (CF-SL) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Astragali Radix (CF-AR) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Glycyrrhizae Radix et Rhizoma (CF-GL) > Carthami Flos-Salviae Miltiorrhizae Radix et Rhizoma (CF-SM) > Carthami Flos (CF), and the contribution degree of ASYB in the safflower and its series herb pairs: CF-GL > CF-PS > CF-AS > CF-SL > CF-SM > CF-AR > CF-GR > CF. So, this study provided a significant and effective approach to elucidate the contribution of different herbal components to the bioactivity of the herb pair, and clarification of the variation of herb-pair compatibilities. In addition, this study provides guidance for investigating the relationship between herbal compounds and the bioactivities of herb pairs. It also provides a scientific basis for reasonable clinical applications and new drug development on the basis of the safflower series of herb pairs.

[Research on nourishing and tonifying blood effects of the herb pair consisting of Angelica sinensis and Ligusticum chuanxiong on the basis of drug interaction].

OBJECTIVE: To study the pharmacodynamic interaction of nourishing and tonifying blood effects of the herb pair consisting of Angelica sinensis and Ligusticum chuanxiong by response surface method. METHODS: The blood deficiency rat model was induced by injecting N-acetylphenylhydrazine and cyclophosphamide. The effects of Angelica sinensis and Ligusticum chuanxiong in different proportions (0:1, 1:5, 2:5, 2:3, 1:1, 3:2, 5:2, 5:1, and 1:0) and at different concentrations on the peripheral blood index and the organ indices were observed. Then all indices were integrated to the total nourishing effect value by comprehensive index method. The interaction was analyzed by response surface method. The model parameters were estimated with nonlinear regression. The three-dimensional response surfaces were constructed with Matlab Software. RESULTS: In the response surface, most compatibility of Angelica sinensis and Ligusticum chuanxiong showed synergistic action, some showed addition action, and few of them showed obvious antagonist action. The proportion of Angelica sinensis and Ligusticum chuanxiong from 4:1 to 2:1 and the dose of Angelica sinensis and Ligusticum chuanxiong from low to high showed addition action, while the other proportions showed obvious addition action at low dose and synergistic action at high dose. CONCLUSIONS: The research results could provide scientific evidence for reasonable application of Angelica sinensis and Ligusticum chuanxiong in clinics of Chinese medicine. The quantitative analysis on drug interactions of herbal compatibility by response surface method could provide reference for relative studies.

[Study on evaluation system for gynecological disease model characterized by Qi stagnation and blood stasis syndrome with female SD rats].

OBJECTIVE: To establish an evaluation system for animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome, in order to disprove syndrome characteristics of the model by classic clinical prescriptions, and evaluate the specificity and reliability of the model with macroscopic biological signs and symptoms. METHOD: The model characterized by Qi stagnation and blood stasis syndrome was established by injecting adrenaline into female SD rats and conducting unpredictable chronic stimulus such as reversal of day and night, swimming in cold water, thermal stimulation in oven, noise and tail suspension for two weeks. They were also orally administered with Xiangfu Siwu Tang, Shaofu Zhuyu Tang and positive control drug aspirin in groups. A comprehensive evaluation was conducted for the model on the basis of haemorheology, four blood coagulation indexes, four diagnostic information (digital imaging of tongue, paw and tail, temperature, weight, ingestion, electrocardiograph, and open filed test), and syndrome rating. RESULT: Compared with the normal group, the model group showed obvious changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, syndrome rating and heart rate. Their tongue and paw pictures were analyzed with Photoshop 7.0, showing significant difference in red, green and blue percentage composition from the normal group. Groups given aspirin and Xiangfu Siwu Tang showed notable changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, heart rate, syndrome rating, and red, green and blue percentage composition in tongue and paw pictures, whereas the group given Shaofu Zhuyu Tang showed no remarkable improvement. CONCLUSION: The evaluation system for the animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome is established to provide reference for studies on the evaluation system for qi stagnation and blood stasis syndrome models.

[Research on Chinese medicine pairs (IV)--Their compatibility effects].

Chinese medicine pairs (CMP) are specific compatibility with traditional Chinese medicine (TCM) theory as principle, which was formed on basis of effect and treatment theory of TCM. The compatibility of CMP can enhance the effect, or reduce the toxicity. So, compatibility effects of CMP were studied from different pharmacology levels including animals, tissues and organs, cell and molecular levels, their synergy and compatibility attenuation were also evaluated with monomer component-component (group) compatibility-single herb-CMP-formulae as the main line, and the action mechanisms were investigated, all of which can provide scientific basis for the clinical applications of CMP and creation of TCM new drugs.

[Interaction of nourishing and tonifying blood effects of the combination of Angelicae sinensis Radix and Astragali Radix studied by response surface method].

The combination of Angelicae sinensis Radix (Danggui, DG) and Astragali Radix (Huangqi, HQ) is a popular herb pair commonly used in clinic for the treatment of blood deficiency syndrome in China. The aim of this paper is to study the interaction of DG and HQ nourishing and tonifying blood effects by response surface method. The blood deficiency mice were induced by injecting N-acetylphenylhydrazine (sc) and cyclophosphamide (ip). The blood deficiency mice were administrated intragastrically with DG-HQ extracts (0:1, 1: 5, 2:5, 2:3, 1:1, 3:2, 5:2, 5:1, 1:0). The changes of the peripheral blood indexes and organ indexes were observed. The indexes were integrated by comprehensive index method; the interactions of DG and HQ were analyzed by the response surface diagram established with Matlab software. The results showed that DG and HQ at most of their combination ratios had synergic effect. Within the range of 1:5 - 5:1, all of the extracts of DG-HQ showed synergic effect, and among which, high-doses had better effects than low-doses. The highest value (-1) of the synergic effect was showed when DG was 10 - 40 g at the same time of HQ as 90 -180 g, and DG was 50 - 100 g at the same time of HQ as 20 - 100 g. DG-HQ at all combination dosages within Chinese Pharmacopeia (DG: 6 - 12 g, HQ: 9 - 30 g) had certain synergic effect, and Danggui Buxue Decoction (DG: 6 g, HQ: 30 g) also was at this range. The results provided scientific basis to the clinical application of DG and HQ. And the response surface method was firstly applied to quantitatively evaluate the bio-activity change of herb combination, which provided a novel way for modern basic research on the interaction of herbs.

Fuzzy identification of bioactive components for different efficacies of rhubarb by the back propagation neural network association analysis of UPLC-Q-TOF/MSE and integrated effects.

BACKGROUND: Rhei Radix et Rhizoma (rhubarb), as one of the typical representatives of multi-effect traditional Chinese medicines (TCMs), has been utilized in the treatment of various diseases due to its multicomponent nature. However, there are few systematic investigations for the corresponding effect of individual components in rhubarb. Hence, we aimed to develop a novel strategy to fuzzily identify bioactive components for different efficacies of rhubarb by the back propagation (BP) neural network association analysis of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry for every data (UPLC-Q-TOF/MSE) and integrated effects. METHODS: Through applying the fuzzy chemical identification, most components of rhubarb were classified into different chemical groups. Meanwhile the integration effect values of different efficacies can be determined by animal experiment evaluation and multi-attribute comprehensive indexes. Then the BP neural network was employed for association analysis of components and different efficacies by correlating the component contents determined from UPLC-Q-TOF/MSE profiling and the integration effect values. Finally, the effect contribution of one type of components may be totaled to demonstrate the universal and individual characters for different efficacies of rhubarb. RESULTS: It suggested that combined anthraquinones, flavanols and their polymers may be the universal character to the multi-functional properties of rhubarb. Other components contributed to the individuality of rhubarb efficacies, including stilbene glycosides, anthranones and their dimers, free anthraquinones, chromones, gallic acid and gallotannins, butyrylbenzenes and their glycosides. CONCLUSIONS: Our findings demonstrated that the bioactive components for different efficacies of rhubarb were not exactly the same and can be systematically differentiated by the network-oriented strategy. These efforts will advance our knowledge and understanding of the bioactive components in rhubarb and provide scientific evidence to support the expansion of its use in clinical applications and the further development of some products based on this medicinal herb.

Studies on blood enrichment and anti-tumor effects of combined Danggui Buxue Decoction, Fe and rhEPO based on colon cancer-related anemia model and gut microbiota modulation.

Colon cancer-related anemia (CCRA) is mainly caused by systemic inflammation, intestinal bleeding, iron deficiency and chemotherapy-induced myelosuppression in colon cancer. However, the best therapeutic schedule and related mechanism on CCRA were still uncertain. Studies on blood enrichment and anti-tumor effects of combined Danggui Buxue Decoction (DBD), Fe and rhEPO based on CCRA and gut microbiota modulation were conducted in this paper. Here, CCRA model was successfully induced by subcutaneous inoculation of CT-26 and i.p. oxaliplatin, rhEPO + DBD high dosage + Fe (EDF) and rhEPO + DBD high dosage (ED) groups had the best blood enrichment effect. Attractively, EDF group also showed antitumor activity. The sequencing results of gut microbiota showed that compared to P group, the relative abundances of Lachnospiraceae and opportunistic pathogen (Odoribacter) in ED and EDF groups were decreased. Interestingly, EDF also decreased the relative abundances of cancer-related bacteria (Helicobacter, Lactococcus, Alloprevotella) and imbalance-inducing bacteria (Escherichia-Shigella and Parabacteroides) and increased the relative abundances of butyrate-producing bacteria (Ruminococcaceae_UCG-014), however, ED showed the opposite effects to EDF, this might be the reason of the smaller tumor volume in EDF group. Our findings proposed the best treatment combination of DBD, rhEPO and Fe in CCRA and provided theoretical basis and literature reference for CCRA-induced intestinal flora disorder and the regulatory mechanism of EDF.

Comparatively Evaluating the Role of Herb Pairs Containing Angelicae Sinensis Radix in Xin-Sheng-Hua Granule by Withdrawal Analysis.

The present study aims to investigate the roles of herb pairs containing Angelicae Sinensis Radix (Danggui) in Xin-Sheng-Hua Granule (XSHG) on hemolytic and aplastic anemia (HAA) mice. HAA model mice were induced by acetyl phenylhydrazine and cyclophosphamide; then the samples of XSHG and its decomposed recipes (DY, DC, DT, DH, DJ, and DZ) were orally administrated to these mice. Indicators of peripheral blood routine, organ index, and ATPase activities were tested. Moreover, the main effective components in these samples were also analyzed by UHPLC-TQ-MS/MS. Clear separation between the control and model groups from score plot of principal component analysis (PCA) was easily seen, indicating that HAA model was successfully conducted. Afterwards, relative distance calculation method between dose groups and control group from PCA score plot was adopted to evaluate the integrated effects of hematinic function of different samples. And the orders of hematinic effects were as follows: XHSG > DJ > DT > DZ > DH > DC > DY. Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicated changes when herb pairs containing Danggui were excluded from XSHG, respectively. Compared with XSHG, the vast majority of active compounds in sample DY (formula minus herb pair Danggui-Yimucao) decreased significantly, which could partly explain why herb pair Danggui-Yimucao made great contribution to XSHG. These findings showed that withdrawal analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG, which could lay foundation to reveal the compatibility rules of this formula.

Integration of organ metabolomics and proteomics in exploring the blood enriching mechanism of Danggui Buxue Decoction in hemorrhagic anemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Decoction (DBD), as a classical Chinese medicine prescription, is composed of Danggui (DG) and Huangqi (HQ) at a ratio of 1:5, and it has been used clinically in treating anemia for hundreds of years. AIM OF THE STUDY: The aim of this study was to explore the treatment mechanisms of DBD in anemia rats from the perspective of thymus and spleen. MATERIALS AND METHODS: In this study, a successful hemorrhagic anemia model was established, and metabolomics (UPLC-QTOF-MS/MS) and proteomics (label-free approach) together with bioinformatics (Gene Ontology analysis and Reactome pathway enrichment), correlation analysis (pearson correlation matrix) and joint pathway analysis (MetaboAnalyst) were employed to discover the underlying mechanisms of DBD. RESULTS: DBD had a significant blood enrichment effect on hemorrhagic anemia rats. Metabolomics and proteomics results showed that DBD regulated a total of 10 metabolites (lysophosphatidylcholines, etc.) and 41 proteins (myeloperoxidase, etc.) in thymus, and 9 metabolites (L-methionine, etc.) and 24 proteins (transferrin, etc.) in spleen. With GO analysis and Reactome pathway enrichment, DBD mainly improved anti-oxidative stress ability of thymocyte and accelerated oxidative phosphorylation to provide ATP for splenocyte. Phenotype key indexes were strongly and positively associated with most of the differential proteins and metabolites, especially nucleosides, amino acids, Fabp4, Decr1 and Ndufs3. 14 pathways in thymus and 9 pathways in spleen were obtained through joint pathway analysis, in addition, the most influential pathway in thymus was arachidonic acid metabolism, while in spleen was the biosynthesis of phenylalanine, tyrosine and tryptophan. Furthermore, DBD was validated to up-regulate Mpo, Hbb and Cp levels and down-regulate Ca2+ level in thymus, as well as up-regulate Fabp4, Ndufs3, Tf, Decr1 and ATP levels in spleen. CONCLUSION: DBD might enhance thymus function mainly by reducing excessive lipid metabolism and intracellular Ca2+ level, and promote ATP production in spleen to provide energy.

An integrated metabolomics strategy to reveal dose-effect relationship and therapeutic mechanisms of different efficacy of rhubarb in constipation rats.

The ambiguity of dose-effect relationship of many traditional Chinese medicines (TCMs) has always influenced their rational use in TCM clinic. Rhubarb, a preferred representative of cathartic TCM, is currently widely used that results in a diversity of its dosage. The aim of this study was to use an integrated metabolomics strategy to simultaneously reveal dose-effect relationship and therapeutic mechanisms of different efficacy of rhubarb in constipation rats. Six doses of rhubarb (0.135, 0.27, 0.81, 1.35, 4.05, and 8.1 g/kg) were examined to elucidate the laxative and fire-purging effects by pathological sections and UPLC-Q-TOF/MSE. The results showed that there existed serious lesions in the stomach and colon of model rats. And conditions were basically improved to some extent in rhubarb-treated groups. Through relative distance calculation based on metabolomics score plots, it suggested that the effective dose threshold (EC20-EC80 range) of rhubarb was from 0.31 to 4.5 g/kg (corresponding to 3.44-50.00 g in the clinic) in rat serum and 0.29-2.1 g/kg (corresponding to 3.22-23.33 g in the clinic) in feces. Then, 33 potential biomarkers were identified in total. Functional pathway analysis revealed that the alterations of these biomarkers were associated with 15 metabolic pathways, mainly including arachidonic acid metabolism, glycerophospholipid metabolism, steroid biosynthesis, primary bile acid biosynthesis and sphingolipid metabolism. Of note, different doses of rhubarb could alleviate endogenous disorders to varying degrees through regulating multiple perturbed pathways to the normal state, which might be in a dose-dependent manner and involved in therapeutic mechanisms. To sum up, integrated serum and fecal metabolomics obtained that rhubarb ranging from 0.31 to 2.1 g/kg is safe and effective for constipation treatment. Also, our findings showed that the robust metabolomics techniques would be promising to be more accurately used in the dose-effect studies of complex TCM, and to clarify syndrome pathogenesis and action mechanisms in Chinese medicine.

Comparative analysis of the main active constituents from different parts of Leonurus japonicus Houtt. and from different regions in China by ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry.

A rapid, sensitive and convenient analytical method of ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (UPLC-QTRAP®/MS2) was proposed for the simultaneous determination of characteristic alkaline and acidic components covering many structure types including alkaloids, phenolic acids, phenylpropanoids and flavonoids in Leonurus japonicus Houtt. (LJ). The proposed method was first reported and validated by assessing the matrix effects, linearity, limit of detections, limit of quantifications, precision, repeatability, stability and recovery of target components. The developed UPLC-QTRAP®/MS2 was successfully applied to simultaneously determine all target compounds in 38 batches of LJ from 11 different producing regions in China and five organs (including root, caulis, branch, flower and leaf) of LJ from the same stand planting base in Jiangsu Province (China). The result showed that LJ in different regions with different geographical position would affect the accumulation of different compounds, and the significant discrepancies of some target compounds were also observed in different organs of LJ due to different biosynthetic pathway and enzymes in different organs. Furthermore, both hierarchical clustering analysis and principal components analysis were performed to classify the 38 batches of LJ samples from different producing regions on the basis of target compounds. As a result, the samples could be mainly clustered into different groups, which were similar with areas classification. Overall, the presented method would be helpful for the comprehensive utilization and development of LJ resources.

A network pharmacology approach to investigate the blood enriching mechanism of Danggui buxue Decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui buxue Decoction (DBD) has been frequently used to treat with blood deficiency, which consisted of Danggui (DG) and Huangqi (HQ) at a ratio of 1:5. Accumulating evidence showed that blood deficiency in traditional Chinese medicine (TCM) was similar to anemia in modern medicine. AIM OF THE STUDY: The purpose of this study was to explore its therapeutic mechanism of with network pharmacology approach. MATERIALS AND METHODS: We explored the chemical compounds of DBD and used compound ADME screening to identify the potential compounds. Targets for the therapeutic actions of DBD were obtained from the PharmMapper, Swiss, SEA and STITCH. GO analysis and pathway enrichment analysis was performed using the DAVID webserver. Cytoscape was used to visualize the compound-target-pathway network for DBD. The pharmacodynamics and crucial targets were also validated. RESULTS: Thirty-six potential active components in DBD and 49 targets which the active components acted on were identified. 47 KEGG pathways which DBD acted on were also come to light. And then, according to KEGG pathway annotation analysis, only 16 pathways seemed to be related to the blood nourishing effect of DBD, such as PI3K-AKT pathway, and so on. Only 32 targets participated in these 16 pathways and they were acted on by 29 of the 36 active compounds. Whole pharmacodynamic experiments showed that DBD had significant effects to blood loss rats. Furthermore, DBD could promote the up-regulation of hematopoietic and immune related targets and the down-regulation of inflammatory related targets. Significantly, with the results of effective rate, molecular docking and experimental validation, we predicted astragaloside IV in HQ, senkyunolide A and senkyunolide K in DG might be the major contributing compounds to DBD's blood enriching effect. CONCLUSION: In this study, a systematical network pharmacology approach was built. Our results provided a basis for the future study of senkyunolide A and senkyunolide K as the blood enriching compounds in DBD. Furthermore, combined network pharmacology with validation experimental results, the nourishing blood effect of DBD might be manifested by the dual mechanism of enhancing immunity and promoting hematopoiesis.