Effects of dietary inclusion of Radix Bupleuri extract on the growth performance, and ultrastructural changes and apoptosis of lung epithelial cells in broilers exposed to atmospheric ammonia

To explore whether Radix Bupleuri extract (RBE) could protect lung injury of broilers under ammonia (NH3) exposure, 360 1-day-old male broilers were randomly allocated to 4 groups of 6 replicates each in a 2 × 2 factorial design with 2 diets (the basal diet (control; CON) and the basal diet supplemented with RBE (RB)) and 2 air conditions (normal condition (< 2 ppm of NH3; NOR) and NH3 exposure (70 ppm of NH3; NH70)). The RB diet contained 80 mg saikosaponins/kg diet. On d 7, the lung tissues were collected and the lung epithelial cells (LEC) were isolated. Our experimental results showed that the NH3 exposure decreased body weight gain and feed intake irrespective of dietary treatments during d 1-7. However, the RBE addition decreased feed consumption to body weight gain ratio in broilers under NH70 condition. In the LEC of CON-fed broilers under NH70 condition, Golgi stacks showed the dilation of cisternaes and reduced secretory vesicles, mitochondria enlarged, the inner membrane of mitochondria became obscure, and the cristae of mitochondria ruptured, whereas only a mild enlargement of Golgi cisternaes and the part rupture of mitochondrial cristaes occurred in the LEC of RB-fed broilers under NH70 condition. The NH3 exposure increased malondialdehyde (MDA) level, but decreased total antioxidant capacity (T-AOC) in the lungs of CON-fed broilers. However, the RBE addition decreased MDA level and increased T-AOC in the lungs of broilers under NH70 condition. Simultaneously, the NH3 exposure increased apoptotic rate (AR), mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) level in the isolated LEC of CON-fed broilers. The RBE addition decreased AR, MMP, and ROS in the isolated LEC of broilers under NH70 condition. Besides, the NH3 exposure increased mRNA expression of B-cell lymphoma-2 associated X protein (BAX), caspase-3, and tumor necrosis factor α (TNF-α), but increased interferon γ (IFN-γ) mRNA abundance in the lungs of CON-fed broilers. The RBE supplement decreased mRNA levels of BAX, caspase-3, and TNF-α, but increased IFN-γ, interleukin (IL)-4, and IL-17 mRNA levels in the lungs of broilers under NH70 condition. These results indicated that dietary RBE addition alleviated NH3 exposure-induced intercellular ultrastructural damage via mitochondrial apoptotic pathway, possibly due to RBE-induced increase of antioxidant capacity and immunomodulatory function in the lungs of broilers under NH3 exposure.

Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS

Saikosaponins comprise a large group of chemical components present in the Bupleurum species that have attracted attention in the field of medicine because of their significant biological activities. Due to the high polarity, structural similarity, and the presence of several isomers of this class of components, their structural identification is extremely challenging. In this study, the mass spectrometric fragmentation pathways, UV spectral features, and chromatographic behavior of different types of saikosaponins were investigated using 24 standard substances. Saikosaponins containing carbonyl groups (C=O) in the aglycone produced fragment ions by loss of 30 Da, and in addition, type IV saikosaponins could produce [aglycone−CH2OH−OH−H]− and [aglycone−H2O−H]− fragment ions through neutral losses at positions C16 and C17. The above characteristic ions can be used to identify saikosaponins. More notably, the identification process of saikosaponins was systematically summarized, and using this method, 109 saikosaponins were identified or tentatively characterized from the saikosaponins extract of Bupleurum marginatum var. stenophyllum (BMS) using UPLC-PDA-Q/TOF-MS with both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, of which 25 were new compounds and 60 were first discovered from BMS. Further studies revealed that the saikosaponins profiles of BMS, Bupleurum chinense DC (BC), and Bupleurum marginatum Wall. ex DC (BMW) were very similar. This work is of great significance for the basic research of the Bupleurum species and provides strong technical support to solve the resource problems associated with Radix Bupleuri.

Comparison and Analysis on the Existing Single-Herbal Strategies against Viral Myocarditis

Purpose. Herbal medicine is one of crucial symbols of Chinese national medicine. Investigation on molecular responses of different herbal strategies against viral myocarditis is immeasurably conducive to targeting drug development in the current international absence of miracle treatment. Methods. Literature retrieval platforms were applied in the collection of existing empirical evidences for viral myocarditis-related single-herbal strategies. SwissTargetPrediction, Metascape, and Discovery Studio coordinating with multidatabases investigated underlying target genes, interactive proteins, and docking molecules in turn. Results. Six single-herbal medicines consisting of Huangqi (Hedysarum Multijugum Maxim), Yuganzi (Phyllanthi Fructus), Kushen (Sophorae Flavescentis Radix), Jianghuang (Curcumaelongae Rhizoma), Chaihu (Radix Bupleuri), and Jixueteng (Spatholobus Suberectus Dunn) meet the requirement. There were 11 overlapped and 73 unique natural components detected in these herbs. SLC6A2, SLC6A4, NOS2, PPARA, PPARG, ACHE, CYP2C19, CYP51A1, and CHRM2 were equally targeted by six herbs and identified as viral myocarditis-associated symbols. MCODE algorithm exposed the hub role of SRC and EGFR in strategies without Jianghuang. Subsequently, we learned intermolecular interactions of herbal components and their targeting heart-tissue-specific CHRM2, FABP3, TNNC1, TNNI3, TNNT2, and SCN5A and cardiac-myocytes-specific IL6, MMP1, and PLAT coupled with viral myocarditis. Ten interactive characteristics such as π-alkyl and van der Waals were modeled in which ARG111, LYS253, ILE114, and VAL11 on cardiac troponin (TNNC1-TNNI3-TNNT2) and ARG208, ASN106, and ALA258 on MMP1 fulfilled potential communicating anchor with ellagic acid, 5α, 9α-dihydroxymatrine, and leachianone g via hydrogen bond and hydrophobic interaction, respectively. Conclusions. The comprehensive outcomes uncover differences and linkages between six herbs against viral myocarditis through component and target analysis, fostering development of drugs.

Studies on the Changes of Pharmacokinetics Behaviors of Phytochemicals and the Influence on Endogenous Metabolites After the Combination of Radix Bupleuri and Radix Paeoniae Alba Based on Multi-Component Pharmacokinetics and Metabolomics

Radix Bupleuri-Radix Paeoniae Alba (RB-RPA) is a classic herb pair, which is commonly used to treat depression by soothing “liver qi stagnation” in the clinic. However, little is yet known concerning the combination mechanism of Radix Bupleuri (RB) and Radix Paeoniae Alba (RPA), their bioactive forms in vivo and the regulatory effects on the organism. The present study aimed to elucidate the changes in multi-component pharmacokinetics (PK) behavior after the combination of RB and RPA by a high-resolution full-scan mode of UPLC-HRMS method (a total of 38 components PK profiles were obtained, of which 23 components come from RB and 15 components come from RPA). Moreover, the metabolomics approach was used to analyze the dynamic response of endogenous metabolites intervened by RB-RPA, and the correlation between concentration-time curves of 38 components from RB-RPA and the dynamic response profiles of endogenous metabolites was characterized by Pearson correlation analysis. The results demonstrated that the combination of RB and RPA could significantly improve the bioavailability of five components in RB, and six components in RPA. Besides, metabolomics results indicated that a total of 21 endogenous metabolites exhibited time-dependent changes in response to the RB-RPA administration, of which 12 endogenous metabolites were significantly increased, and nine endogenous metabolites were significantly decreased. Furthermore, correlation analysis results indicated that the components with significantly improved bioavailability after combination such as saikogenin F, saikogenin G, albiflorin, methyl gallate, paeonimetabolin II were significantly positively correlated with picolinic acid, a metabolite with neuroprotective effect; saikogenin F, saikogenin G were significantly positively correlated with itaconic acid, a endogenous metabolite with anti-inflammatory activity; and albiflorin, paeonimetabolin II were significantly positively correlated with α-linolenic acid, a metabolite with strong protective actions on brain functions. These results indicated that the combination of RB and RPA can enhance each other’s neuroprotective and anti-inflammatory activities. In this study, A novel and efficient strategy has been developed to analyze the influence of the combination of RB and RPA in vivo behaviors by combining multi-component pharmacokinetics with metabolomics, which was contributed to clarifying the scientific connotation of herb–herb compatibility.

Saikosaponin D Rescues Deficits in Sexual Behavior and Ameliorates Neurological Dysfunction in Mice Exposed to Chronic Mild Stress

Often associated with sexual dysfunction (SD), chronic stress is the main contributing risk factor for the pathogenesis of depression. Radix bupleuri had been widely used in traditional Chinese medicine formulation for the regulation of emotion and sexual activity. As the main active component of Radix bupleuri, saikosaponin D (SSD) has a demonstrated antidepressant effect in preclinical studies. Herein, we sought to investigate the effect of SSD to restore sexual functions in chronically stressed mice and elucidate the potential brain mechanisms that might underly these effects. SSD was gavage administered for three weeks during the induction of chronic mild stress (CMS), and its effects on emotional and sexual behaviors in CMS mice were observed. The medial posterodorsal amygdala (MePD) was speculated to be involved in the manifestation of sexual dysfunctions in CMS mice. Our results revealed that SSD not only alleviated CMS-induced depressive-like behaviors but also rescued CMS-induced low sexual motivation and poor sexual performance. CMS destroyed astrocytes and activated microglia in the MePD. SSD treatment reversed the changes in glial pathology and inhibited neuroinflammatory and oxidative stress in the MePD of CMS mice. The neuronal morphological and functional deficits in the MePD were also alleviated by SSD administration. Our results provide insights into the central mechanisms involving the brain associated with sexual dysfunction. These findings deepen our understanding of SSD in light of the psychopharmacology of stress and sexual disorders, providing a theoretical basis for its potential clinical application.

Sulfation of Quercitrin, Epicatechin and Rutin by Human Cytosolic Sulfotransferases (SULTs): Differential Effects of SULT Genetic Polymorphisms

Radix Bupleuri is one of the most widely used herbal medicines in China for the treatment of fever, pain, and/or chronic inflammation. Quercitrin, epicatechin, and rutin, the flavonoids present in Radix Bupleuri, have been reported to display anti-inflammatory, antitumor, and antioxidant biological activities among others. Sulfation has been reported to play an important role in the metabolism of flavonoids. In this study, we aimed to systematically identify the human cytosolic sulfotransferase enzymes that are capable of catalyzing the sulfation of quercitrin, epicatechin, and rutin. Of the thirteen known human cytosolic sulfotransferases, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1C2, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward quercitrin, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A3, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward epicatechin, and six (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A2, cytosolic sulfotransferase 1A3, cytosolic sulfotransferase 1B1, cytosolic sulfotransferase 1C4, and cytosolic sulfotransferase 1E1) displayed sulfating activity toward rutin. The kinetic parameters of the cytosolic sulfotransferases that showed the strongest sulfating activities were determined. To investigate the effects of genetic polymorphisms on the sulfation of quercitrin, epicatechin, and rutin, individual panels of cytosolic sulfotransferase allozymes previously prepared were analyzed and shown to display differential sulfating activities toward each of the three flavonoids. Taken together, these results provided a biochemical basis underlying the metabolism of quercitrin, epicatechin, and rutin through sulfation in humans.

Synergistic Effect of Saikosaponin a and Albiflorin Against Corticosterone-Induced Neuro-Injury in PC12 Cells via Regulation of TCA Cycle, Purine Metabolism, and Glutamate Metabolism

The combination of Radix Bupleuri - Radix Paeoniae alba is one of the most approbated herb pairs in traditional Chinese medicine (TCM) formula for curing depression. Saikosaponin A (SSA) and albiflorin (AF) are major bioactive compounds of Radix Bupleuri and Radix Paeoniae alba respectively, which possess antidepressant effects in pharmacological experiments. However, whether SSA and AF have synergistic neuroprotective effects and the synergistic mechanism is still unknown. The present study employed the corticosterone-induced PC12 cells neuro-injury model to assess the protective effect of SSA and AF, alone and in combination, and the synergistic effect was analyzed using three mathematical models. Meanwhile, the cell metabolomics was used to detect the effects on metabolite regulation of SSA and AF, alone and in combination. According to the results of cell metabolomics, the vital cell transduction pathway related to the crucial cell metabolic pathways were selected. Furthermore, the key metabolites, metabolic enzymes, and cellular markers were verified by ELISA and Western blotting. The results showed that the combination of SSA and AF has a synergistic neuroprotective effect. Besides, the combination could regulate more metabolites (16) than a single agent (12 & 10) and possessed a stronger adjustment effect on metabolites. Correspondingly, the combination regulated more metabolism pathways (7) than alone agents (3 & 5). Furthermore, the TCA cycle, purine metabolism, and glutamate metabolism were selected as crucial metabolism pathways. The results showed that the TCA cycle disorder was regulated by SSA and AF via improving mitochondrial function. The purine metabolism disorder was regulated by SSA via inhibition xanthine oxidase (XOD) activity and the glutamate metabolism disorder was regulated by AF via inhibition glutaminase (GLS) activity. Moreover, the oxidative stress induced by the purine metabolism disorder was attenuated by SSA via a reduction in the ROS level. Additionally, the inflammation induced by the oxidative stress was attenuated by the SSA and AF via inhibition of the NLRP3 protein expression. This study for the first time demonstrated the synergistic neuroprotective effect of SSA and AF, and the synergistic mechanism was involved in metabolic disorders regulation.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum

Background: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. Objective: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements on SS biosynthesis. Method: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. Results: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton β-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. Conclusion: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide reference for maintaining the stability of SS content in Radix Bupleuri.