Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

Structure-activity relationship and biofilm formation-related gene targets of oleanolic acid-type saponins from Pulsatilla chinensis against Candida albicans.

In the course of our investigations of antifungal natural products, the structure-activity relationship and antifungal activities of oleanolic acid-type saponins (1-28) from Pulsatilla chinensis against human and plant pathogenic fungi were elucidated. The analysis of structure-activity relationship of oleanolic acid-type saponins showed that the free carboxyl at C-28 was essential for their antifungal activities; the free hydroxyl group at the C-23 site of oleanolic acid-type saponins played a crucial role in their antifungal activities; the oligosaccharide chain at C-3 oleanolic acid-type saponins showed significant effects on antifungal efficacy and a disaccharide or trisaccharide moiety at position C-3 displayed optimal antifungal activity. The typical saponin pulchinenoside B3 (16, PB3) displayed satisfactory antifungal activity against human and plant pathogenic fungi, especially, C. albicans with an MIC value of 12.5 µg/mL. Furthermore, PB3 could inhibit the biofilm formation of C. albicans through downregulating the expression of the integrated network of biofilm formation-associated transcription factors (Bcr1 Efg1, Ndt80, Brg1, Rob1 and Tec1) and adhesion-related target genes (HWP1, ALS1, and ALS3). Meanwhile, we found that PB3 could effectively destroy the mature biofilm of C. albicans by the oxidative damage and inducing mitochondria-mediated apoptosis in cells.

Effect of the Pulsatilla decoction n-butanol extract on vulvovaginal candidiasis caused by Candida glabrata and on its virulence factors.

Vulvovaginal candidiasis (VVC) caused by Candida glabrata (C. glabrata) is more persistent and resistant to treatment than when caused by Candida albicans (C. albicans) and has been on the rise in recent years. The n-butanol extract of Pulsatilla Decoction (BEPD) has been shown to be effective in treating VVC caused by C. glabrata, but the underlying mechanism of action remains unclear. In this study, the experimenter conducted in vitro and in vivo experiments to explore the effects of BEPD on the virulence factors of C. glabrata, as well as its efficacy, with a focus on possible immunological mechanism in VVC caused by C. glabrata. The contents of Anemoside B4, Epiberberine, Berberine, Aesculin, Aesculetin, Phellodendrine and Jatrorrhizine in BEPD, detected by high-performance liquid chromatography, were 31,736.64, 13,529.66, 105,143.72, 19,406.20, 4952.67, 10,317.03, 2489.93 µg/g, respectively. In vitro experiments indicated that BEPD moderately inhibited the growth of C. glabrata, its adhesion, and biofilm formation, and affected the expression of efflux transporters in the biofilm state. In vivo experiments demonstrated that BEPD significantly reduced vaginal inflammatory manifestation and the release of proinflammatory cytokines and LDH in mice with VVC caused by C. glabrata. Moreover, it inhibited the Phosphorylation of EGFR, ERK, P38, P65, and C-Fos proteins. The results suggested that although BEPD moderately inhibits the growth and virulence factors of C. glabrata in vitro, it can significantly reduce vaginal inflammation by down-regulating the EGFR/MAPK signaling pathway in mice with VVC infected by C. glabrata.

A Comprehensive Review on Botany, Phytochemistry, Traditional Uses, Pharmacology, Analytical Methods, Processing Methods, Pharmacokinetics and Toxicity of Pulsatilla chinensis.

Objective: To lay the foundation for further research and clinical application of Pulsatilla chinensi (Bge.) Regel (PR). Method: The information presented in this paper is based on scientific databases (such as Baidu Academic, CNKI, Google Academic, PubMed, Science Direct, Web of Science, SciFinder Scholar), traditional Chinese medicine classics, Chinese Pharmacopoeia, doctoral and master's dissertations, etc. Various compounds were identified and isolated through HPLC analysis, including glycosides, polysaccharides, organic acids, phenylpropane, and flavonoids. Results: These compounds have demonstrated a range of pharmacological effects such as anti-cancer, anti-inflammatory, insecticidal, immune-enhancing, antimutagenic, antioxidant, antiviral, antibacterial, hepatoprotective, and antidiarrheal properties. Its chemical composition and pharmacological effects are complex and diverse. Conclusion: There is a need for further studies to investigate its mechanism, potential coordination, and antagonism.

Mechanism underlying the effect of Pulsatilla decoction in hepatocellular carcinoma treatment: a network pharmacology and in vitro analysis.

BACKGROUND: Currently, hepatocellular carcinoma (HCC) is associated with a poor prognosis. Moreover, there exist limited strategies for treating HCC. Pulsatilla decoction (PD), a traditional Chinese medicine formula, has been used to treat inflammatory bowel disease and several cancer types. Accordingly, we explored the mechanism of PD in HCC treatment via network pharmacology and in vitro experiments. METHODS: Online databases were searched for gene data, active components, and potential target genes associated with HCC development. Subsequently, bioinformatics analysis was performed using protein-protein interaction and Network Construction and Kyoto Encyclopedia of Genes and Genomes (KEGG) to screen for potential anticancer components and therapeutic targets of PD. Finally, the effect of PD on HCC was further verified by in vitro experiments. RESULTS: Network pharmacological analysis revealed that 65 compounds and 180 possible target genes were associated with the effect of PD on HCC. These included PI3K, AKT, NF-κB, FOS, and NFKBIA. KEGG analysis demonstrated that PD exerted its effect on HCC mainly via the PI3K-AKT, IL-17, and TNF signaling pathways. Cell viability and cell cycle experiments revealed that PD could significantly inhibit cancer cell proliferation and kill HCC cells by inducing apoptosis. Furthermore, western blotting confirmed that apoptosis was mediated primarily via the PI3K-AKT, IL-17, and TNF signaling pathways. CONCLUSION: To the best of our knowledge, this is the first study to elucidate the molecular mechanism and potential targets of PD in the treatment of HCC using network pharmacology.

The effect of plant compartment and geographical location on shaping microbiome of Pulsatilla chinensis.

The plant-associated microbiome has an effect on plant growth. Pulsatilla chinensis (Bge.) Regel is an important Chinese medicinal plant. Currently, there is little understanding of the P. chinensis-associated microbiome and its diversity and composition. Here, the core microbiome associated with the root, leaf, and rhizospheric soil compartments of P. chinensis from five geographical locations was analyzed by the metagenomics approach. The alpha and beta diversity analysis showed that the microbiome associated with P. chinensis was shaped by the compartment, especially in the bacterial community. The geographical location had little influence on microbial community diversity associated with root and leaf. Hierarchical clustering distinguished the microbial communities of rhizospheric soil based on their geographical location and among the soil properties, pH was showed the more stronger effect on the diversity of rhizospheric soil microbial communities. Proteobacteria was the most dominant bacterial phylum in the root, leaf, and rhizospheric soil. Ascomycota and Basidiomycota were the most dominant fungal phyla in different compartments. Rhizobacter, Anoxybacillus, and IMCC26256 were the most important marker bacterial species for root, leaf, and rhizospheric soil screened by random forest, respectively. The fungal marker species for root, leaf, and rhizospheric soil were not only different across the compartments but also the geographical locations. Functional analysis showed that P. chinensis-associated microbiome had the similar function which had no obvious relationship with geographical location and compartment. The associated microbiome indicated in this study can be used for identifying microorganisms related to the quality and growth of P. chinensis. KEY POINTS: • Microbiome associated with P. chinensis was shaped by the compartment • Microbiome composition and abundance associated with rhizospheric soil were affected by the geographical location • Compared with fungi, bacterial associated with P. chinensis composition and diversity were more stable in different geographical locations and compartments.

Qualitative and quantitative analysis of triterpenoids in different tissues of Pulsatilla chinensis.

Pulsatilla chinensis (P.chinensis) is a traditional Chinese medicine used for the treatment of intestinal amebiasis diseases, vaginal trichomoniasis and bacterial infections. Tritepenoid saponins were important components of P.chinensis. Therefore, we asssessmented expression profiling of triterpenoids in different fresh tissues of P.chinensis by ultra high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS). Firstly, we identified 132 triterpenoids, including 119 triterpenoid saponins, 13 triterpenoid acids and forty seven of them were first determined in Pulsatilla genus, including new aglycones and new ways of rhamnose linking to the aglycone. Secondly, we established the analytical method to analysis triterpenoids content of P.chinensis and comprehensively verified the analytical method by linearity, precision, repeatability, stability and recovery. At last, we quantified 119 triterpenoids simultaneously based on UHPLC-QQQ-MS. The results show that the types and contents of triterpenoids had obvious tissue distribution. New components like rhamnose directly linked to the aglycone mainely distributed in aboveground tissues. Additionally, We identified 15 chemical ingredients as differential components between the aboveground and underground tissues of P.chinensis. This study provides an efficient analysis strategy for the qualitative and quantitative analysis of triterpenoids in P.chinensis even in other traditional Chinese medicines. At the same time, it provides important informations to explain the biosynthetic pathway of triterpenoid saponins in P.chinensis.

Pulsatilla Saponins Inhibit Experimental Lung Metastasis of Melanoma via Targeting STAT6-Mediated M2 Macrophages Polarization.

Pulsatilla saponins (PS) extracts from Pulsatilla chinensis (Bge.) Regel, are a commonly used traditional Chinese medicine. In the previous study, we found Pulsatilla saponins displayed anti-tumor activity without side effects such as bone marrow suppression. However, the mechanism of the anti-tumor effect was not illustrated well. Since M2-like tumor-associated macrophages (TAMs) that required activation of the signal transducer and activator of transcription 6 (STAT6) for polarization are the important immune cells in the tumor microenvironment and play a key role in tumor progress and metastasis, this study aimed to confirm whether Pulsatilla saponins could inhibit the development and metastasis of tumors by inhibiting the polarization of M2 macrophages. We investigated the relevance of M2 macrophage polarization and the anti-tumor effects of Pulsatilla saponins in vitro and in vivo. In vitro, Pulsatilla saponins could decrease the mRNA level of M2 marker genes Arg1, Fizz1, Ym1, and CD206, and the down-regulation effect of phosphorylated STAT6 induced by IL-4; moreover, the conditioned medium (CM) from bone marrow-derived macrophages (BMDM) treated with Pulsatilla saponins could inhibit the proliferation and migration of B16-F0 cells. In vivo, Pulsatilla saponins could reduce the number of lung metastasis loci, down-regulate the expression of M2 marker genes, and suppress the expression of phosphorylated STAT6 in tumor tissues. Furthermore, we used AS1517499 (AS), a STAT6 inhibitor, to verify the role of PS on M2 macrophage polarization both in vitro and in vivo. We found that Pulsatilla saponins failed to further inhibit STAT6 activation; the mRNA level of Arg1, Fizz1, Ym1, and CD206; and the proliferation and migration of B16-F0 cells after AS1517499 intervention in vitro. Similar results were obtained in vivo. These results illustrated that Pulsatilla saponins could effectively suppress tumor progress by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway; this revealed a novel mechanism for its anti-tumor activity.

Pulsatilla chinensis saponins ameliorated murine depression by inhibiting intestinal inflammation mediated IDO1 overexpression and rebalancing tryptophan metabolism.

BACKGROUND: Current antidepressant therapy remains unsatisfactory due to the complex pathogenesis. Emerging evidence suggested that depression is associated with inflammatory bowel disease (IBD), intestinal inflammation is an increasingly accepted factor that influences depression, but the mechanism is unclear. PURPOSE: In the current study, we determined whether Pulsatilla chinensis saponins (PRS), a phytomedicine from Pulsatilla chinensis (Bunge) Regel with excellent anti-IBD effect, could improve the depression. Furthermore, we investigated the mechanisms to explore the relationship between IBD and depression and provide new source for the urgent development of antidepressants from phytomedicine. METHODS: The antidepressant activity of PRS was accessed by behavioral test and multichannel technology in depression mice induced by Chronic Unpredictable Mild Stress (CUMS). 16S rDNA-based microbiota and RNA-seq in colon was used to explore potential intestinal metabolism affected by PRS. To illustrate the underlying mechanisms of anti-depression effect of PRS, targeted metabolomics, ELISA assay, immunofluorescence staining, Western Blot, and qPCR were carried out. RESULTS: The results clarified that CUMS induced depression with tryptophan (Trp) metabolism and intestinal inflammation. PRS effectively suppressed the depression and acted as a regulator of Trp/kynurenine (Kyn) metabolic and intestinal inflammation confirmed by analysis of microflora and colon RNA. Meanwhile PRS reduced interferon gamma (IFN-γ), inhibited JAK1-STAT1 phosphorylation, decreased IDO1 levels to protect against the overactivity of Trp/kyn path, suggesting that IFN-γ activated IDO1 probably a significant target for PRS to exert anti-depression effects. To further confirm the mechanism, this research expressed that PRS improved IDO1 activity and depressive behavior in mice with IFN-γ-induced depression. Furthermore, the therapeutic effect of 1-methyl-tryptophan (1-MT) well known as an IDO1 inhibitor in depression and clinically used anti-UC drug Mesalazine (MS) was demonstrated to confirm the potential mechanism. CONCLUSION: The study is the first to reveal the antidepressant effect of PRS and further demonstrate its potential therapeutic targets. In addition, it also clarifies that the Trp/kyn pathway is the crosstalk between IBD and depression and provides new choice for depression treatment. And it also provides an important basis for the follow-up development and exploration of anti-intestinal antidepressants.

Therapeutic effects of Pulsatilla koreana Nakai extract on ovalbumin-induced allergic rhinitis by inhibition of Th2 cell activation and differentiation via the IL-4/STAT6/GATA3 pathway.

Allergic rhinitis (AR), caused by immunoglobulin E (IgE)-mediated inflammation, generally occurs in the upper respiratory tract. T helper type 2 (Th2) cell-mediated cytokines, including interleukin (IL)-4, IL-5, and IL-13, are important factors in AR pathogenesis. Despite various treatment options, the difficulty in alleviating AR and pharmacological side effects necessitate development of new therapies. The root of Pulsatilla koreana Nakai (P. koreana), a pasque flower, has been used as a herbal medicine. However, its effects on AR remain unclear; therefore, we aimed to explore this subject in the current study. The therapeutic effects of P. koreana water extract (PKN) on the pathophysiological functions of the nasal mucosa was examined in ovalbumin (OVA)-induced AR mice. The effect of PKN on Th2 activation and differentiation was evaluated using concanavalin A-induced splenocytes and differentiated Th2 cells from naïve CD4+ T cells. We also investigated the effect of changes in JAK/STAT6/GATA3 signaling on IL-4-induced Th2 cells. In OVA-induced AR mice, PKN administration alleviated allergic nasal symptoms and decreased the total number of immune cells, lymphocytes, neutrophils, and eosinophils in nasal lavage fluid; serum levels of OVA-specific IgE, histamine, and IL-13 were also significantly reduced. PKN also ameliorated OVA-induced nasal mucosal tissue thickening by inhibiting inflammation and goblet cell hyperplasia. PKN treatment significantly inhibited Th2 activity and differentiation through the IL-4/STAT-6/GATA3 pathway in Th2 cells. PKN is an effective AR treatment with the potential to improve patients' daily lives by regulating the allergic inflammatory response induced by Th2 cells.

Computational method for rapid screening of the metabolites of Pulsatilla chinensis in rats using UHPLC-Q-TOF/MS combined with mass spectrum-based orthogonal projection.

Screening metabolites in vivo can be challenging due to the complexity of traditional Chinese medicine (TCM) and the ambiguous intracorporal process. To resolve this problem, we established the mass spectrum-based orthogonal projection (MSOP) method to differentiate prototype compounds from metabolites in vivo and applied it to the study of metabolites of Pulsatilla chinensis (PC). Initially, the validity and feasibility of the MSOP method were verified by using the ultra- high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) data of reference solution. Then, the MSOP method was applied to screen the metabolites of PC. A total of 63 metabolites were identified in vivo (urine, feces, bile, and plasma samples) and in vitro (intestinal bacteria biological sample). The results indicated that the main metabolic pathways of pentacyclic triterpenoids were demethylation, oxidation, dehydration, sulfation, and glucuronidation reactions. This study contributes to developing an integrated strategy based on chemometrics to characterize and classify the metabolism feature of pentacyclic triterpenoids of PC. This will support the scientific and rational application of PC in the clinic. The MSOP method based on the orthogonality of MS signals was used to differentiate the prototype compounds from metabolites in vivo. The method provides scientific and reliable support for fully understanding the metabolic fate of TCM.

Pulsatilla chinensis saponins improve SCFAs regulating GPR43-NLRP3 signaling pathway in the treatment of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction has been extensively used to treat ulcerative colitis (UC) in recent years. Pulsatilla chinensis saponin (PRS), the active ingredient of its monarch medicine Pulsatilla chinensis (Bunge) Regel, plays a crucial role in the treatment of UC, but its specific mechanism of action has not been fully elucidated. AIM OF THE STUDY: This study aims to investigate the protective effect and possible mechanism of PRS on DSS-induced ulcerative colitis in rats. MATERIALS AND METHODS: In this study, the DSS-induced colitis model was used to explore the metabolism and absorption of PRS under UC, detect the content of short-chain fatty acids (SCFAs) in colon tissue, the expression of receptor G Protein-Coupled Receptor 43 (GPR43) protein and inflammasome NLRP3, and observe the expression level of IL-1ß, IL-6 and TNF-α in colon tissue. The protective effect of the PRS was also observed. RESULTS: It was found that in the UC group, the absorption rate and extent of drugs increased, and the elimination was accelerated. Compared with the control group, PRS increased the content of short-chain fatty acids (SCFAs) in colon tissue, promoted the expression of SCFAs receptor GPR43 protein, inhibited the activation of the NLRP3 inflammasome, and decreased the content of IL-1ß, IL-6 and TNF-α. PRS protects the colon in DSS-induced inflammatory bowel disease by increasing the content of SCFAs, promoting the expression of GPR43 protein, inhibiting the activation of the NLRP3 inflammasome, and reversing the increase in IL-1ß, IL-6 and TNF-α levels. CONCLUSIONS: PRS can increase the content of colonic SCFAs, activate the GPR43-NLRP3 signaling pathway, and reduce the levels of pro-inflammatory cytokines, thereby improving the symptoms of DSS-induced colitis.

A Case of Recurrent Urinary Tract Infection Successfully Treated during Homeopathic Supportive Care in Oncology.

INTRODUCTION: A 32-year-old patient with colon cancer consulted for homeopathic supportive care (HSC). She had also suffered from recurrent urinary tract infections (RUTIs) for 20 years. Could homeopathy treat these two very different issues with the same medicine? THE PATIENT'S MAIN CONCERNS: Though the main reason for the consultation was a fear of the side effects of chemotherapy, the presence of Escherichia coli-induced RUTIs affected the patient's quality of life. INTERVENTIONS AND OUTCOMES: The repertory listing highlighted Pulsatilla as the patient's homeopathic constitutional medicine. It was prescribed both for HSC and also for RUTI. In association with Arsenicum album and Nerves, Pulsatilla enabled a good tolerance to chemotherapy, with rapid recovery from peripheral neuropathies. In combination with Colibacillinum, Pulsatilla provided relapse-free curing of the RUTIs. The MOdified NARanjo Criteria for Homeopathy (MONARCH) Inventory score was +9. CONCLUSION: The one individual's two different clinical complaints were treated with a constitutional homeopathic medicine, Pulsatilla, which covered the whole case. However, in HSC, the use of the constitutional remedy alone is rarely sufficient: it was reinforced by individualised symptomatic medication, organotherapy and isotherapy. For RUTI, isopathic and tubercular miasmatic treatments were each helpful. For both complaints, individualised homeopathy gave rapid, long-lasting and effective results.

Discovery, Preliminary Structure-Activity Relationship, and Evaluation of Oleanane-Type Saponins from Pulsatilla chinensis for the Treatment of Ulcerative Colitis.

To discover ulcerative colitis (UC) treatment agents, 28 oleanane-type triterpenoid saponins (1-28) including three new saponins, pulsatillosides P-R (1-3), were isolated from Pulsatilla chinensis. The isolated saponins could observably ameliorate UC by improving the intestinal epithelial cell barrier and intestinal flora in vivo. The structure-activity relationship indicated that the oligosaccharide chain at C-28 was essential for their anti-UC activities; the methyl group at the C-23 site of triterpene saponins showed important effects on anti-UC efficacy; the chain length of oligosaccharides at position C-28 had little effect on their anti-UC activities. In vivo investigation of representative saponins 1 and 13 further confirmed that 23-methyl-3,28-bisdesmosidic oleanane-type saponins inhibited the TNFα-NFκB-MLCK axis to improve the intestinal epithelial cell barrier of the colon in UC mice. These findings revealed the potential of 23-methyl-3,28-bisdesmosidic oleanane-type saponins from P. chinensis as promising candidates for the treatment of UC.

Pulsatilla vulgaris Inhibits Cancer Proliferation in Signaling Pathways of 12 Reporter Genes.

This study aimed to examine if methanolic extracts of Pulsatilla vulgaris Mill. can inhibit HeLa cell proliferation through the modulation of cancer-related signaling pathways. The cytotoxicity and chemical composition of P. vulgaris leaves and root extracts were also determined. Research showed that root extract of P. vulgaris inhibited 12 signaling pathways in a cervical cancer cell line and the most potent activation inhibition was observed for MYC, Notch, Wnt, E2F, Ets, Stat3, Smad, Hdghog, AP-1, and NF-κB, at a concentration of 40 µg/mL. The methanolic extracts of P. vulgaris enhanced apoptotic death and deregulated cellular proliferation, differentiation, and progression toward the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of P. vulgaris on cancer signaling pathways. Additionally, our detailed phytochemical analysis of the methanolic extracts of P. vulgaris gives a conclusion that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC. AIM OF THE STUDY: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1ß and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. RESULTS: After administration of BEPD-containing serum, the levels of IL-1ß, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. CONCLUSIONS: BEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

Pulsatilla decoction improves DSS-induced colitis via modulation of fecal-bacteria-related short-chain fatty acids and intestinal barrier integrity.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction (PD), is an herbal formula commonly used for the treatment of ulcerative colitis (UC) in clinical practice, but the mechanism of PD alters the colitis remains elusive. AIM OF THE STUDY: To evaluate the intervention effect of PD on Dextran Sodium Sulfate (DSS)-induced UC based on gut microbiota and intestinal short-chain fatty acid (SCFAs) metabolism, and to investigate the mechanism of action of PD in treating UC. MATERIALS AND METHODS: A 3% (wt/vol) DSS-induced ulcerative colitis model in C57BL/6 male mice was used to evaluate the effect of oral PD in treating UC. The changes in gut microbiota in mice were analyzed by 16SrDNA gene sequencing, and the content of SCFAs in the intestinal contents of mice was determined by gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was applied to analyze the expression of inflammatory cytokines in serum and colonic tissues, and western blotting (WB) was applied to analyze the expression of tight junction proteins in colonic tissues. RESULTS: PD can alleviate the symptoms of UC mice, Pulsatilla Decoction high dose treatment group (PDHT) shows the best effect. Compared with the DSS group, the PDHT had significantly lower body mass, disease activity index (DAI) score, colonic macroscopic damage index (CMDI) score, and pathological damage score, at the phylum level, the relative abundance of Bacteroidetes increased while that of Firmicutes and Proteobacteria decreased, at the Genus level, the abundance of Bacteroides and Lachnospiraceae.NK4A136.group increased while that of Clostridium. sensu.stricto。, Escherichia. shigella and Turicibacter decreased. Compared with the DSS group, acetate, propionate, and total SCFAs in the PDHT with significantly higher levels. The concentrations of interleukin-1ß (L-1ß), tumor necrosis factor-alpha (TNF-α), and interleukin-17 (IL-17) decreased whereby the concentration of interleukin-10 (IL-10) increased in the PDHT group. The expression levels of Occludin, zonula occludens-1 (ZO-1), Claudin1, Claudin5, G protein-coupled receptor43 (GPR43) protein, and the relative expression of ZO-1 and Occludin mRNA were significantly increased PDHT group. CONCLUSIONS: PD has a good therapeutic effect on UC mice. The pharmacological mechanism is probably maintaining the homeostasis and diversity of gut microbiota, increasing the content of SCFAs, and repairing the colonic mucosal barrier.

Study on a novel enzymatic colon-targeted particle of total saponins of Pulsatilla by mechanical grinding technology in a solvent free system.

Oral colon-targeting preparation can achieve targeted drug release in the lesion site and have a great application prospect. In this study, we presented the principle of particle design in the field of materials science into the preparation of colon-targeting preparation. Enzymatic Pulsatilla saponins Colon-targeting composite Microparticles (EPCM) were prepared by mechanical grinding without any organic solvent. Firstly, Response Surface Methodology (RSM) designed by Box-Behnken was used to optimize the preparation process of EPCM, and the structure of EPCM was characterized. All-Atomic and Molecular Dynamics (AAMD) was used to calculate the compatibility between drugs and coating materials before and after release, so as to explain the principle of colon- targeted drug release in this method. Then, colon-targeting characteristics of EPCM in vitro and in vivo were investigated by experiments. Finally, the pharmacodynamics effects of this composite microparticles on Ulcerative Colitis (UC) induced by DSS in C57BL/6 mice were evaluated. The results demonstrated that the colon-targeting composite microparticles could be prepared by mechanical grinding based on particle design principle. The composite microparticles have appropriate colon-targeting drug release performance in vitro and in vivo, and have good anti-ulcerative colitis effect. This study provides a new idea for the preparation of oral colon-targeting preparation of Traditional Chinese medicine, and has evident reference value for the study of coating isolation and powder modification of traditional Chinese medicine for other purposes.

Isolation and characterisation of Pulsatilla Radix-utilising bacteria Pediococcus pentosaceus PR-1 from human faeces.

Although probiotics have been isolated from different sources, few were isolated from traditional Chinese medicine. The current study firstly isolates Pulsatilla Radix-utilising Pediococcus pentosaceus PR-1 from human faeces. Subsequently, the tolerance of PR-1 to low pH, bile salts, simulated gastric juice and succus entericus, antioxidant activity, antimicrobial activity, cholesterol assimilation and antibiotics susceptibility were investigated. After 2 h of incubation at pH 2.0, over 80% of PR-1 survived. The cell viability of PR-1 at 2 h under 0.1% bile salt condition was 99.2%. The survival rate of PR-1 in gastric juice and succus entericus was 64.48% and 81.86%, respectively. Cell-free supernatant of PR-1 culture also showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. Besides, antioxidant activity of PR-1 CFS was significantly greater than cell pellet. PR-1 was shown to be resistant to kanamycin, streptomycin, vancomycin and norfloxacin and was able to lower the cholesterol level to 72.5±1.5%. In addition, PR-1 displayed γ-haemolysis and was non-pathogenic.

Pulsatilla decoction suppresses matrix metalloproteinase-7-mediated leukocyte recruitment in dextran sulfate sodium-induced colitis mouse model.

BACKGROUND: Intestinal inflammation is considered to be an important characteristic of ulcerative colitis (UC) and the current medical treatments for UC are usually proposed to suppress abnormal intestinal immune responses. Pulsatilla decoction (PD), a traditional Chinese medicine, is frequently used in UC treatments in Asian countries; however, the mechanism of the action of PD remains unclear. In the present study, the mechanism of the action of PD was elucidated in the dextran sulfate sodium (DSS)-induced colitis mouse model, a model to mimic UC. METHODS: Murine colitis was evaluated by comparing the disease activity index score. The intestinal inflammation was examined by histology analyses. The leukocyte infiltration in the colonic tissues was examined by immunohistochemistry analyses. The cytokines level in colonic tissues was examined by Multi-Plex immunoassay. The epithelial proliferation was evaluated by histological analyses. Immunofluorescence double staining was used to examine the expression of MMP-7 in the immune cells. RESULTS: In the DSS-induced colitis mouse model, administration of PD attenuated the intestinal inflammation, with a marked decrease in colonic infiltration of innate immune cells. Immunohistochemical analyses further showed that matrix metalloproteinase-7 (MMP-7) expressed by the infiltrating leukocytes, including neutrophils and macrophages was inhibited by PD treatment. PD increases the cytokine level of IL-6 in colonic tissues. CONCLUSION: PD suppresses intestinal inflammation, with a marked decrease in colonic infiltration of innate immune cells, through decreasing MMP-7 expression.