[Mechanism of protective effect of n-butanol extract of Pulsatilla Decoction on vaginal epithelial cells under Candida albicans stimulation through EGFR/MAPK pathway based on transcriptomics].

This study aimed to investigate the protective effect and its underlying mechanism of n-butanol extract of Pulsatilla Decoction(BEPD) containing medicinal serum on vaginal epithelial cells under Candida glabrata stimulation via the epidermal growth factor receptor/mitogen activated protein kinase( EGFR/MAPK) pathway based on transcriptomics. A vulvovaginal candidiasis(VVC) mouse model was established first and transcriptome sequencing was performed for the vaginal mucosa tissues to analyze the gene expression differences among the control, VVC model, and BEPD intervention groups. Simultaneously, BEPD-containing serum and fluconazole-containing serum were prepared. A431 cells were divided into the control, model, blank serum, fluconazole-containing serum, BEPD-containing serum, EGFR agonist and EGFR inhibitor groups. Additionally, in vitro experiments were conducted using BEPD-containing serum, fluconazole-containing serum, and an EGFR agonist and inhibitor to investigate the intervention mechanisms of BEPD on C. glabrata-induced vaginal epithelial cell damage. Cell counting kit-8(CCK-8) assay was utilized to determine the safe concentrations of C. glabrata, drug-containing serum, and compounds on A431 cells. Enzyme-linked immunosorbent assay(ELISA)was employed to measure the expression levels of interleukin(IL)-1ß, IL-6, granulocyte-macrophage colony-stimulating factor(GMCSF), granulocyte CSF(G-CSF), chemokine(C-X-C motif) ligand 20(CCL20), and lactate dehydrogenase(LDH). Gram staining was used to evaluate the adhesion of C. glabrata to vaginal epithelial cells. Flow cytometry was utilized to assess the effect of C.glabrata on A431 cell apoptosis. Based on the transcriptomics results, immunofluorescence was performed to measure the expressions of p-EGFR and p-ERK1/2 proteins, while Western blot validated the expressions of p-EGFR, p-ERK1/2, p-C-Fos, p-P38, Bax and Bcl-2 proteins. Sequencing results showed that compared with the VVC model, BEPD treatment up-regulated 1 075 genes and downregulated 927 genes, mainly enriched in immune-inflammatory pathways, including MAPK. Mechanistically, BEPD significantly reduced the expression of p-EGFR, p-ERK1/2, p-C-Fos and p-P38, as well as the secretion of IL-1ß, IL-6, GM-CSF, G-CSF and CCL20, LDH release induced by C. glabrata, and the adhesion of C. glabrata to A431 cells, suggesting that BEPD exerts a protective effect on vaginal epithelial cells damaged by C. glabrata infection by modulating the EGFR/MAPK axis. In addition, BEPD downregulated the pro-apoptotic protein Bax expression and up-regulated the anti-apoptotic protein Bcl-2 expression, leading to a reduction in C. glabrata-induced cell apoptosis. In conclusion, this study reveals that the intervention of BEPD in C. glabrata-induced VVC may be attributed to its regulation of the EGFR/MAPK pathway, which protects vaginal epithelial cells.

[Mechanism of n-butanol extract of Pulsatilla Decoction in treating ulcerative colitis by activating BMP signaling pathway].

The study aimed to investigate the therapeutic effects of the n-butanol extract of Pulsatilla Decoction(BEPD) on ulcerative colitis(UC) via the bone morphogenetic protein(BMP) signaling pathway. C57BL/6 mice were divided into six groups: control, model, mesalazine, and BEPD low-, medium-, and high-dose groups. Except for the control group, the rest groups were treated with 3% dextran sulfate sodium(DSS) freely for seven consecutive days to establish the UC mouse model, followed by treatment with different concentrations of BEPD and mesalazine by gavage. The murine body weight and disease activity index(DAI) were recorded. After the mice were sacrificed, their colon tissues were collected for histological analysis. Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to detect the number and mucus secretion status of goblet cells; immunohistochemistry was performed to measure the expression of ki67, cleaved caspase-3, mucin 2(Muc2), and matrix metalloproteinase-9(MMP9) in colon tissues; and immunofluorescence was used to analyze the expression of tight junction proteins in colon tissues, and enzyme linked immunosorbent assay(ELISA) was employed to quantify the levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1ß, and IL-6. Western blot was conducted to evaluate the expression of BMP pathway-related proteins in mouse colon tissues. Quantitative real-time PCR(qRT-PCR) was performed to measure the expression of genes related to goblet cell differentiation in mouse colon tissues. In addition, this study also examined the protective effect and underlying mechanism of BEPD-containing serum on lipopolysaccharide(LPS)-induced barrier damages in LS174T goblet cells in vitro. The results showed that BEPD significantly alleviated UC symptoms in mice, restored goblet cell diffe-rentiation function, promoted Muc2 secretion and tight junction protein expression, and suppressed inflammatory factor secretion while activating the BMP signaling pathway. Therefore, BEPD may exert its therapeutic effects on UC by activating the BMP signaling pathway, providing a new strategy for drug intervention in UC.

Effect of Pulsatilla decoction on vulvovaginal candidiasis in mice. Evidences for its mechanisms of action.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common infection that affects the female reproductive tract. Pulsatilla decoction (PD), a traditional Chinese herbal medicine, is a classic and effective prescription for VVC. However, its mechanism of action remains unclear. PURPOSE: This study aimed to evaluate the efficacy and potential mechanism of action of the n-butanol extract of Pulsatilla decoction (BEPD) in VVC treatment. METHODS: High performance liquid chromatography (HPLC) was used to detect the main active ingredients in BEPD. A VVC-mouse model was constructed using an estrogen-dependent method to evaluate the efficacy of BEPD in VVC treatment. Fungal burden and morphology in the vaginal cavity were comprehensively assessed. Candida albicans-induced inflammation was examined in vivo and in vitro. The effects of BEPD on the Protein kinase Cδ (PKCδ) /NLR family CARD domain-containing protein 4 (NLRC4)/Interleukin-1 receptor antagonist (IL-1Ra) axis were analyzed using by immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and reverse transcription-quantitative polymerase chain reaction (qRT-PCR). RESULTS: BEPD inhibited fungal growth in the vagina of VVC mice, preserved the integrity of the vaginal mucosa, and suppressed inflammatory responses. Most importantly, BEPD activated the "silent" PKCδ/NLRC4/IL-1Ra axis and negatively regulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thereby exerting a therapeutic efficacy on VVC. CONCLUSIONS: BEPD effects on mice with VVC were dose-dependent. BEPD protects against VVC by inhibiting inflammatory response and NLRP3 inflammasome via the activation of the PKCδ/NLRC4/IL-1Ra axis. This study revealed the pharmacological mechanism of BEPD in VVC treatment and provided further evidence for the application of BEPD in VVC treatment.

Identification of Pulsatilla chinensis (Bge.) Regel and look-alike species by UHPLC-TOF-MS using multivariate statistical analysis.

Pulsatillae Radix, the root of Pulsatilla chinensis (Bge.) Regel, is recorded in the Pharmacopoeia of the People's Republic of China and has been widely used for its pharmacological activities, such as anti-inflammatory, antioxidant, antibacterial, antitumor, and cardiovascular benefits. However, there are several look-alike species that can be marketed as Pulsatillae Radix. To distinguish P. chinensis (Bge.) Regel from its look-alikes, viz. Pulsatilla cernua (Thunb.) Bercht et Opiz., Pulsatilla dahurica (Fisch.) Spreng., Anemone tomeutosa (Maxim.) Pei., and Rhaponticum uniflorum (L.) DC, we used ultra high performance liquid chromatography with time-of-flight mass spectrometry combined with principal component analysis to compare their chemical compositions. Four ions, a (RT 8.98 min, m/z 1381.6671), b (RT 10.64 min, m/z 1219.6143), c (RT 11.52 min, m/z 1217.5978), and d (RT 13.6 min, m/z 749.4463), from P. chinensis (Bge.) Regel were identified as potential chemical markers to distinguish it from look-alike species using an unsupervised statistical model combined with orthogonal partial least-squares discriminant analysis. The results of this study provide an effective method for identifying and distinguishing P. chinensis (Bge.) Regel from similar plants.

Pulsatilla Decoction Combined with 5-Fluorouracil Triggers Immunogenic Cell Death in Colorectal Cancer Cells.

Background: Our research is designed to explore the role of 5-FU and Pulsatilla decoction (PD) through modulation of Immunogenic cell death (ICD) for the co-treatment of Colorectal cancer (CRC). Materials and Methods: Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assays. Cell apoptosis was assessed using flow cytometry. Phosphorylation of STAT3 and expression of Mcl-1 and Bcl-xl were measured by Western blot assays. The levels of ATP and HMGB1 in the supernatants of the culture medium were analyzed by ATP assays and the HMGB1 enzyme linked immunosorbent assay kit. The cell surface levels of CRT were measured by immunofluorescence assays. The tumor growth was analyzed in mice. Results: PD increased 5-FU-induced ICD in CRC cells, as demonstrated by the extracellular levels of adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), and the surface levels of calreticulin (CRT). Our mechanism study showed that PD promoted 5-FU-induced ICD by inactivating signal transducer and activator of transcription 3 (STAT3). Furthermore, the co-treatment of 5-FU and PD further promoted 5-FU-induced CRT expression and T cell infiltration in vivo. Tumorigenicity analysis revealed that 5-FU combined with PD notably reduced tumor growth. Conclusion: This study indicated that PD enhances 5-FU-induced ICD and anti-tumor effect in CRC by inactivating STAT3. The combined application of 5-FU with PD may improve the anti-tumor activity of 5-FU in CRC.

Extracts from Pulsatilla patens target cancer-related signaling pathways in HeLa cells.

The purpose of this study was to determine if a methanolic extract of the Pulsatilla patens (L.) Mill. can inhibit the progression of cancer through the modulation of cancer-related metabolic signaling pathways. We analyzed a panel of 13 inducible luciferase reporter gene vectors which expression is driven by enhancer elements that bind to specific transcription factors for the evaluation of the activity of cancer signaling pathways. The root extract of P. patens exhibited strong inhibition of several signaling pathways in HeLa cells, a cervical cancer cell line, and was found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog, at a concentration of 40 µg/mL. The methanolic extracts of P. patens enhanced apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Further, our detailed phytochemical analysis of the methanolic extracts of the P. patens allowed to deduce that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.

AB4 inhibits Notch signaling and promotes cancer cell apoptosis in liver cancer.

The etiology for liver cancer has been clearly defined. Unfortunately, therapeutic approaches for liver cancer are rather limited, and liver cancer is insensitive to chemotherapy and radiotherapy. Traditional Chinese medicine (TCM) has become a promising strategy for cancer treatment as TCM elicits broad spectrum anticancer activity. In the present study, we evaluated the anticancer efficacy of AB4, an extract from the medical herb Pulsatilla chinensis (Bunge) Regel, in liver cancer in vitro and in vivo. We found that AB4 readily dose­ and time­dependently inhibited liver cancer HepG2 and Huh­7 cell proliferation and colony formation. Western blot and flow cytometry analyses suggested that AB4 treatment induced liver cancer cell apoptosis. Moreover, these findings could be readily recaptured in vivo, in which the AB4 regimen resulted in tumor suppression and cancer cell apoptosis in xenograft tumor­bearing nude mice. Importantly, we noted that treatment with a Notch signaling inhibitor DAPT produced very similar anticancer efficacy in both HepG2 and Huh­7 cell lines, and administration of DAPT also efficiently suppressed HepG2 xenograft outgrowth. To this end, we anticipated that AB4 and DAPT may act on the same signaling pathway, probably through inhibition of the Notch pathway. Indeed, we found decreased expression of Notch1 protein, as well as downstream targets Hes1 and Hey1, after AB4 treatment. Immunohistochemistry analysis further confirmed the suppression of Notch signaling in HepG2 xenograft­bearing mice. Taken together, our study highlighted the anticancer efficacy of AB4 in liver cancer. We also provided preliminary data showing Notch as a therapeutic target of AB4. It would be interesting to investigate the anticancer efficacy of AB4 in other types of cancer with elevated Notch activity.

Identification of cytochrome P450 isoenzymes involved in the metabolism of 23-hydroxybetulinic acid in human liver microsomes.

Context: 23-Hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), exhibits potential antitumor activity. Its metabolism, however, has not yet been studied.Objective: This study focuses on the metabolism of 23-HBA in vitro by human liver microsomes.Materials and methods: The metabolic kinetics of 23-HBA (0.5-100 µM) and the effects of selective CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) inhibitors on metabolism of 23-HBA were evaluated in human liver microsomes incubation system and then determined by LC-MS method. The Michaelis-Menten parameters Km and Vmax were initially estimated by analysing Lineweaver-Burk plot. The clearance (CLint) was also calculated.Results: The Vmax, Km, and CLint of 23-HBA were 256.41 ± 11.20 pmol/min/mg, 11.10 ± 1.07 µM, and 23.10 ± 1.32 µL/min/mg, respectively. The metabolism of 23-HBA was significantly inhibited by furafylline (0.05 µM, p < 0.01) and ketoconazole (0.02 µM, p < 0.05). Ticlopidine (1.3 µM, p < 0.05) could inhibit the metabolism of 23-HBA, while the other inhibitors (sulfaphenazole and quinidine) showed nonsignificant inhibition on the metabolism of 23-HBA.Discussion and conclusions: This is the first investigation of the metabolism of 23-HBA in human liver microsomes. The in vitro study indicates that CYP1A2 and CYP3A4 are mainly involved in the metabolism of 23-HBA. Special attention should be given to the pharmacokinetic and clinical outcomes when 23-HBA was co-administrated with other compounds mainly undergoing CYP1A2/CYP3A4-mediated metabolism. Further studies are needed to evaluate the significance of this interaction and strengthen the understanding of traditional Chinese medicine.

SB365, Pulsatilla Saponin D Induces Caspase-Independent Cell Death and Augments the Anticancer Effect of Temozolomide in Glioblastoma Multiforme Cells.

SB365, a saponin D extracted from the roots of Pulsatilla koreana, has been reported to show cytotoxicity in several cancer cell lines. We investigated the effects of SB365 on U87-MG and T98G glioblastoma multiforme (GBM) cells, and its efficacy in combination with temozolomide for treating GBM. SB365 exerted a cytotoxic effect on GBM cells not by inducing apoptosis, as in other cancer cell lines, but by triggering caspase-independent cell death. Inhibition of autophagic flux and neutralization of the lysosomal pH occurred rapidly after application of SB365, followed by deterioration of mitochondrial membrane potential. A cathepsin B inhibitor and N-acetyl cysteine, an antioxidant, partially recovered cell death induced by SB365. SB365 in combination with temozolomide exerted an additive cytotoxic effect in vitro and in vivo. In conclusion, SB365 inhibits autophagic flux and induces caspase-independent cell death in GBM cells in a manner involving cathepsin B and mainly reactive oxygen species, and its use in combination with temozolomide shows promise for the treatment of GBM.

Anti-inflammatory and immune-modulatory properties of anemoside B4 isolated from Pulsatilla chinensis in vivo.

BACKGROUND: Pulsatilla chinensis is commonly used for the treatment of cancers and inflammatory disorders in China. Our recent studies showed that anemoside B4, its major ingredient, possessed notable antioxidant and protected cisplatin-induced acute kidney injury in vivo. Furthermore, we found the protective effect might be involved its anti-inflammation activities. However, its anti-inflammatory mechanisms are not clear. PURPOSE: In the present study, we extensively investigated the anti-inflammatory and immune-modulatory properties of anemoside B4 in vivo. METHODS: To carry out this work, the xylene-induced ear edema and LPS-induced systemic inflammation of mice model was also used to evaluate the anti-inflammatory activity. Then, anti-inflammatory mechanism of anemoside B4 was further determined by pro-inflammatory cytokines production using enzyme-linked immunosorbent assay (ELISA) and nuclear factor-κ-gene binding (NF-κB) pathway activation by Western blot. At last, immuno-modulatory effects were observed by splenocyte proliferation assay, delayed type hypersensitivity assay (DTH) and T cell subtype assay in mice. RESULTS: 12.5-50 mg/kg anemoside B4 significantly suppressed xylene-induced mice ear edema. Furthermore, it ameliorated LPS-induced kidney and lung inflammation damage, which inhibited pro-inflammatory response by NF-κB pathway in mice. In addition, anemoside B4 decreased CD4+/CD8+ ratio, inhibited splenic lymphocyte proliferation and decreased DNFB-induced changes of ear thickness. CONCLUSION: From these data, it can be concluded that anemoside B4 presented anti-inflammatory and immune-modulatory activities in vivo, and potentially be a novel natural anti-inflammatory drug candidate for treating inflammatory disorder.

Hederacolchiside C inhibits Enterovirus 71 propagation through activating innate immunity.

Enterovirus 71 (EV71), a newly emerging life-threatening pathogen induces hand-foot-mouth disease (HFMD), no effective vaccines or specific anti-viral treatments are currently available. In this study, the activity of hederacolchiside C (HSC) against EV71 was investigated, and the antiviral mechanism was explored. HSC displayed apparent antiviral activity in EV71-infected cells probably through activating the host innate immunity. Comparing with EV71-infected group at 24 hpi, the group pretreated with HSC dramatically increased the expression of MAVS, p-IRF3, IRF3 and IFN-ß, the innate immune effectors related to innate immunity. In addition, HSC displayed stronger antiviral activity in EV71-infected suckling mice in comparison with Ribavirin, a broad-spectrum antiviral drug. The results suggest that HSC could have potential as a pharmaceutical drug for HFMD.

Safety investigation of Pulsatilla chinensis saponins from chronic metabonomic study of serum biomedical changes in oral treated rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla chinensis (Bunge) Regel is a valuable traditional Chinese medicine (TCM) which is widely used for the treatment of schistosomiasis, inflammatory, bacterial infections. In recent years, P chinensis has been reported to exhibit antitumor activities. However, the mechanisms underlying its toxic effects remain largely unresolved. This paper is designed to investigate the damage of long-term oral P. chinensis saponins (PRS) and to explore its potential damage mechanisms by serum metabonomics approach. MATERIALS AND METHODS: The serum samples from control and PRS treated rats were analyzed by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in positive ionization mode and negative ionization mode. Liver function index of ALT, AST and ALP, blood biochemistry and biomarkers were examined to identify specific changes of injury. Acquired data were subjected to principal component analysis (PCA) for differentiating the control and PRS treated groups. Then, serum metabolic profiling was analyzed and pathway analysis performed on the biomarkers reversed after PRS treated and further integration of metabolic networks. RESULTS: The results suggested that serum liver function indexes of ALT had significantly changed and stage increased. AST, ALP detection content show volatility changes. Changes in the 15 biomarkers found in the serum, such as acetaminophen glucuronide, 9 E, 11 E-linoleic acid, chenodeoxycholic acid, monoacylglycerides, sphingomyelin (SM), 7-ketodeoxycholic acid and 12-keto-deoxycholic acid, which were closely related to changes in liver injury. It could be seen clearly that with the change of the dosing time, the biomarkers in the serum have undergone obvious, regular and progressive changes through the score plot and corresponding loading plot. The underlying regulations of PRS-perturbed metabolic pathways were discussed according to the identified metabolites. CONCLUSION: The present study proves the potential of UPLC-QTOF-MS based metabonomics in mapping metabolic response. Long-term oral administration of P. chinensis saponins can cause chronic liver injury, and its safety needs further attention. It is of great significance in safeguarding human health to explore the damage mechanism of Pulsatilla chinensis saponins on liver by serum metabolomics.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Antischistosomal Properties of Hederacolchiside A1 Isolated from Pulsatilla chinensis.

Background: Schistosomiasis is a major neglected disease for which the current control strategy involves mass treatment with praziquantel, the only available drug. Hence, there is an urgent need to develop new antischistosomal compounds. Methods: The antischistosomal activity of hederacolchiside A1 (HSA) were determined by total or female worm burden reductions in mice harboring Schistosoma japonicum or S. mansoni. Pathology parameters were detected on HSA against 1-day-old S. japonicum-harboring mice. Moreover, we confirmed the antischistosomal effect of HSA on newly transformed schistosomula (NTS) of S. japonicum in vitro. Results: HSA, a natural product isolated from Pulsatilla chinensis (Bunge) Regel, was initially corroborated to possess promising antischistosomal properties. We demonstrated that HSA had high activity against S. japonicum and S. mansoni less in 11 days old parasites harbored in mice. The antischistosomal effect was even more than the currently used drugs, praziquantel, and artesunate. Furthermore, HSA could ameliorate the pathology parameters in mice harboring 1-day-old juvenile S. japonicum. We also confirmed that HSA-mediated antischistosomal activity is partly due to the morphological changes in the tegument system when NTS are exposed to HSA. Conclusions: HSA may have great potential to be an antischistosomal agent for further research.

UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis to identify triterpenoid saponins.

INTRODUCTION: Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE: To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY: In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS: As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION: This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

A sensitive HPLC-MS/MS method for the simultaneous determination of anemoside B4, anemoside A3 and 23-hydroxybetulinic acid: Application to the pharmacokinetics and liver distribution of Pulsatilla chinensis saponins.

Pulsatilla chinensis saponins, the major active components in the herb, have drawn great attention as potential hepatitis B virus infection and hepatoma treatments. Here, a sensitive and accurate HPLC-MS/MS method was established for simultaneous determination of three saponins - anemoside B4, anemoside A3 and 23-hydroxybetulinic acid - in rat plasma and liver, and fully validated. The method was successfully applied to a pharmacokinetics and liver distribution study of P. chinensis saponins. Consequently, 23-hydroxybetulinic acid, with an extremely low content in the P. chinensis saponins, exhibited the highest exposure in the liver and in sites before and after hepatic disposition, namely, in the portal vein plasma and systemic plasma, followed by anemoside B4, which showed the highest content in the herb, whereas anemoside A3 displayed quite limited exposure. The hepatic first-pass effects were 71% for 23-hydroxybetulinic acid, 27% for anemoside B4 and 37% for anemoside A3, corresponding to their different extents of liver distribution. To our knowledge, this is the first investigation on the liver first-pass effect and distribution of P. chinensis saponins to date. These results also provide valuable information for the understanding of the pharmacological effect of P. chinensis saponins on liver diseases.

The derivatives of Pulsatilla saponin A, a bioactive compound from Pulsatilla chinensis: Their synthesis, cytotoxicity, haemolytic toxicity and mechanism of action.

The strong haemolytic toxicity of Pulsatilla saponin A (PSA) has hampered its clinical development as an injectable anticancer agent. To circumvent this challenge, twenty PSA derivatives with C ring or C-28 or C-3 modifications were synthesized and evaluated for cytotoxicity against seven selected human tumor lines, as well as for haemolytic toxicity. Structure-activity relationship (SAR) and structure-toxicity relationship (STR) correlations were also elucidated. Compared with PSA, compound 22 showed a better balance between haemolytic toxicity (HD50 > 500 µM) and cytotoxicity toward lung cancer cells A549 (IC50 = 4.68 µM). Molecular studies indicated that 22 was liked to lead to G1 cell cycle arrest and therefore, 22 may be a potent antitumor drug candidate.

Rapid Detection and Characterisation of Triterpene Saponins from the Root of Pulsatilla chinensis (Bunge) Regel by HPLC-ESI-QTOF-MS/MS.

INTRODUCTION: Triterpene saponins are the major bioactive components in the root of Pulsatilla chinensis (Bunge) Regel (RPC), and have been reported to possess antitumor and immunological adjuvant activities. However, the isolation, purification and elucidation procedures of triterpene saponins from RPC are difficult and time consuming due to high polarity and structural similarity. OBJECTIVES: To develop an analytical strategy for discovering and elucidating triterpene saponins in RPC. METHODS: Methanolic extract of RPC is analysed by high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight-mass spectrometry (HPLC-ESI-QTOF-MS/MS). The MS and MS/MS experiments are conducted using the negative-ionisation mode, in order to provide molecular-mass information and production spectra for the structural elucidation of compounds. RESULTS: Based on retention times, accurate mass and mass spectrometric fragmentation, 24 triterpene saponins are identified or tentatively elucidated from RPC, of which nine triterpene saponins were not reported previously. CONCLUSION: The HPLC-ESI-QTOF-MS/MS could be employed as a rapid, effective technique to screen and identify triterpene saponins in RPC without tedious and time-consuming isolation of pure constituents. Copyright © 2016 John Wiley & Sons, Ltd.

[Pulsatilla decoction inhibits vulvovaginal Candida albicans proliferation and reduces inflammatory cytokine levels in vulvovaginal candidiasis mice].

OBJECTIVE: To explore the possible regulatory effect of Pulsatilla decoction on Th17 cells and inflammatory cytokines of vulvovaginal candidiasis (VVC) mice. METHODS: Seventy-two female Kunming mice were randomly assigned into six groups: a blank control group, a VVC model group, a fluconazole group and three Pulsatilla decoction groups (dose levels: 22.5, 15.0 and 7.5 g/kg, respectively). The VVC mouse models were established by vaginal inoculation with Candida albicans (C. albicans) in female mice in pseudoestrus state caused by estradiol injection. After 7-day treatment on VVC mice, the vaginal C. albicans burden was assessed using dilution spread plate method; the vaginal C. albicans morphology was observed by Gram staining method; the levels of interleukin 6 (IL-6), IL-17, IL-21 and tumor necrosis factor α (TNF-α) in sera were detected by ELISA. The content of the transcription factor retinoid related orphan receptor gamma t (RORγt) in vaginal tissues was detected by immunohistochemistry. RESULTS: The VVC mouse models were successfully developed. After treatment, the vaginal C. albicans burden of the fluconazole group and 22.5 g/kg Pulsatilla decoction group dropped significantly compared with that of the VVC model group. Gram staining showed that the VVC mice had lots of C. albicans hyphae in vaginal discharge, that 7.5 g/kg Pulsatilla decoction group remained the mycelia-phase C. albicans, and that 15.0 g/kg Pulsatilla decoction group had the majority of yeast-phase C. albicans and a few of mycelia-phase, while no hyphae and only very few of yeast-phase C. albicans were observed in 22.5 g/kg Pulsatilla decoction group and fluconazole group. After 7-day treatment, compared with the model group, the levels of IL-6, IL- 17, IL-21 and TNF-α in the sera of the fluconazole group, 15.0 and 22.5 g/kg Pulsatilla decoction groups were reduced significantly and the levels of RORγt in the vaginal tissues of the fluconazole group, 15.0 and 22.5 g/kg Pulsatilla decoction groups also decreased significantly. CONCLUSION: Pulsatilla decoction could inhibit the proliferation of vulvovaginal C. albicans and reduces the levels of inflammatory cytokines in VVC mice.