Digital identification and adulteration analysis of Pulsatilla Radix and Pulsatilla Cernua based on "digital identity" and UHPLC-QTOF-MSE.

Under the background of digitalization of traditional Chinese medicine (TCM), to realize the quick identification and adulteration analysis of Pulsatilla Radix (PR), adhering to digital conviction, this study conducted UHPLC-QTOF-MSE analysis on PR and its adulterant-Pulsatilla Cernua (PC) from different batches and based on digital conversion, the shared ions were extracted from different batches of PR and PC as their "ions representation", respectively. Further, the data set of unique ions of PR relative to PC and PC relative to PR were screened out as the "digital identities" of PR and PC respectively. Further, above the "digital identities" of PR and PC were used as the benchmarks for matching and identifying to feedback give a matching credibility (MC). The results showed that based on the "digital identities" of PR and PC, the digital identification of two herbal samples can be realized efficiently and accurately at the individual level with the MC≥70.00 %, even if 5 % of PC in the mixed samples can still be identified efficiently and accurately. The study is of great practical significance for improving the identification efficiency of PR and PC, cracking down on adulterated and counterfeit drugs, and strengthening the quality control of PR. In addition, it has important reference significance for developing non-targeted digital identification of herbal medicines at the individual level based on UHPLC-QTOF-MSE and the "digital identity", which was beneficial to the construction of digital Chinese medicine and digital quality control.

[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.

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.

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.

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.

Study on the mechanism of reducing biofilm toxicity and increasing antioxidant activity in vinegar processing phytomedicines containing pentacyclic triterpenoid saponins.

ETHNOPHARMACOLOGICAL RELEVANCE: Pentacyclic triterpenoid saponin (PTS) is a kind of particular chemicals with various pharmacological activities, as well as surface activity, mucosal irritation and hemolysis. PTS is closely related to the exertion of efficacy or adverse reactions in plant medicines rich in this component. For the better clinical application of natural resources, how to reduce toxicity and enhance curative efficacy is an important problem which needs to be solved at present. Till now, there has been few studies directly investigating the problem. AIM OF STUDY: Through comparison study of Radix Bupleuri (Chai hu) and Pulsatilla chinensis (Bai tou weng), which are typical traditional Chinese medicines containing PTS, explore the potential change rule of material basis and the mechanism of detoxification and synergistic effect of vinegar processing. MATERIALS AND METHODS: Composition change rule after vinegar processing was applied by UPLC-QTOF-MS/MS coupled with principal component analysis (PCA). Based on our previous research, this paper expounded the action mechanism from the perspective of reducing biofilm toxicity and increasing antioxidant activity. Direct toxicity reducing information was obtained at the cellular level including cellular morphology, MTT assays, western blots and RT-PCR in L02 cells with overload sphingomyelin (SM). The synergistic effect was investigated through histological examinations, mesenteric hemorheology, ELISA, flow cytometry and confocal microscopy. RESULTS: It was found that the structure of PTS take place a series of chemical reactions in the process of vinegar processing which enabled the more toxic components transformed into less toxic components and components with clear efficacy, so as to achieve the purpose of detoxification and synergistic effect. The results indicated that the mechanism of detoxification in vinegar processing was that vinegar processing could act on SM, cause less balance disturbance to sphingomyelin/ceramide (SM/Cer), inhibit apoptosis and then alleviate toxicity. In addition, the pharmacodynamic results showed that the vinegar processing could have an obvious synergistic effect through anti-oxidant stress. CONCLUSIONS: By changing the structures of the PTS, the SM/Cer disrupt was reduced and the antioxidant activity was enhanced, so as to decrease toxicity and increase efficiency in vinegar processing phytomedicines containing PTS.

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.

Therapeutic potential of triterpenoid saponin anemoside B4 from Pulsatilla chinensis.

Pulsatilla Decoction (Bai-Tou-Weng-Tang) has been used medically in China for thousands of years for the treatment of diseases caused by bacteria. In recent decades, Pulsatilla Decoction is becoming a well-known formula prescription used for the treatment of ulcerative colitis in traditional Chinese medicine. Pulsatilla chinensis is the chief herbal source of Pulsatilla Decoction, and it is rich in triterpenoid saponins, such as anemoside B4, anemoside A3, and 23-hydroxybetulinic acid. Anemoside B4 is the most abundant of that group and has been used as a quality control marker for Pulsatilla chinensis. As the major active component of Pulsatilla chinensis, anemoside B4 has also received attention as a pure compound for its therapeutic potential. In this review, we systematically analyze the findings on triterpenoid saponins, especially anemoside B4, anemoside A3 and 23-hydroxybetulinic acid, included in Pulsatilla chinensis and Pulsatilla Decoction. We discuss the pharmacokinetics and tissue distribution of these triterpenoid saponins as well as their biological activities. We also summarize the pharmacological effects of anemoside B4 and its two possible metabolites, anemoside A3 and 23-hydroxybetulinic acid, as pure compounds. In summary, this review sketches a profile of the state of existing knowledge with regard to the pharmacological effects of anemoside B4, especially its anti-inflammatory and immunomodulatory effects. These findings point to the possibility that anemoside B4 has potential to be studied further as a natural compound-originated immunomodulatory agent for the treatment of inflammatory diseases such as ulcerative colitis and thus, may represent one of the most important active components of Pulsatilla Decoction responsible for its anti-ulcerative colitis efficacy.

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.

A new triterpenoid saponin from Pulsatilla cernua predicted by NMR-based mosaic method.

A saponin (1) with gypsogenin as aglycone was isolated from the roots of Pulsatilla cernua. The aglycone of compound 1 was considered as gypsogenin which was rarely found in this genus. Its structure was predicted by NMR-based "mosaic" method rapidly, and further confirmed on the basis of spectroscopic data, including 2D NMR spectra and chemical evidence. This work suggested that NMR-based mosaic method is suitable for most of saponins from common species of genus Pulsatilla.

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.

Flavonoids isolated from the flowers of Pulsatilla flavescens and their anti-piroplasm activity.

Pulsatilla species are known as "Yargui", and their flowers are traditionally used in Mongolia as a tonic and for the treatment of inflammatory diseases. By chemical investigation of P. flavescens flowers, 21 flavonoids, including a new chalcone C-glucoside, chalconaringenin 2'-O-ß-D-glucopyranosyl-5'-ß-D-glucopyranoside, and two new flavanone C-glucosides, (2R)- and (2S)-naringenin 8-ß-D-glucopyranosyl-4'-O-ß-D-glucopyranoside, were isolated. The absolute configurations of the seven flavanone glucosides were elucidated by ECD spectra. For the isolated compounds, inhibitory activity against Babesia caballi and Theileria equi, which cause fatal diseases in horses, was estimated. Although most of the isolated chalcone and flavanone derivatives did not show any anti-piroplasm activity, all the isolated flavone and flavonol derivatives showed moderate effects against B. caballi and/or T. equi.

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.