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ObjectiveFrom the perspective of colorectal cancer stem cells，the purpose of this study was to explore the effect and its mechanism of Spica Prunellae on the proliferation and stemness of colorectal cancer cells based on Notch signaling pathway.MethodsColorectal cancer SW480 cells were cultured in vitro. When the cells were in logarithmic growth phase, they were randomly divided into control group, 0.1 mg/mL group, 0.2 mg/mL group and 0.4 mg/mL group. The control group did not receive drug intervention; the other three groups were added with 0.1 mg/mL, 0.2 mg/mL and 0.4 mg/mL Spica Prunellae solution, respectively. Meanwhile, DMSO in each group was supplemented to the same amount. After 24 hours of intervention, the subsequent experiments were performed. The cell growth morphology and density were observed by inverted microscope. The number of viable cells was detected by 0.4% Trypan blue staining and cell survival rate was calculated. The proliferation inhibition rate of colorectal cancer cells was detected using Cell Counting Kit-8 (CCK8) assay. Flow cytometry was used to detect the proportion of stem cells in colorectal cancer cells. The self-renewal ability of colorectal cancer cells was detected by sphere formation assay. Western blot method was used to detect the relative expression of c-Myc, CD44, CD133, SOX2 and Notch pathway-related proteins Notch1 and Hes1 in colorectal cancer cells.ResultsCompared with the control group, the proliferation and viability of colorectal cancer SW480 cells in the 0.1, 0.2, and 0.4 mg/mL groups were significantly inhibited with the increase of Spica Prunellae concentration (PPPP

The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae. [ABSTRACT FROM AUTHOR]

Abstract Background Thyroid-associated ophthalmopathy (TAO) is an autoimmune inflammatory disorder, which lacks effective treatment currently. Spica Prunellae (SP) is popularly used for its anti-inflammatory and immune-regulating properties, indicating SP may have potential therapeutic value in TAO. Therefore, the purpose of this study is to identify the efficiency and potential mechanism of SP in treating TAO. Methods A network pharmacology integrated molecular docking strategy was used to predict the underlying molecular mechanism of treating TAO. Firstly, the active compounds of SP were obtained from TCMSP database and literature research. Then we collected the putative targets of SP and TAO based on multi-sources databases to generate networks. Network topology analysis, GO and KEGG pathway enrichment analysis were performed to screen the key targets and mechanism. Furthermore, molecular docking simulation provided an assessment tool for verifying drug and target binding. Results Our results showed that 8 targets (PTGS2, MAPK3, AKT1, TNF, MAPK1, CASP3, IL6, MMP9) were recognized as key therapeutic targets with excellent binding affinity after network analysis and molecular docking-based virtual screening. The results of enrichment analysis suggested that the underlying mechanism was mainly focused on the biological processes and pathways associated with immune inflammation, proliferation, and apoptosis. Notably, the key pathway was considered as the PI3K-AKT signaling pathway. Conclusion In summary, the present study elucidates that SP may suppress inflammation and proliferation and promote apoptosis through the PI3K-AKT pathway, which makes SP a potential treatment against TAO. And this study offers new reference points for future experimental research and provides a scientific basis for more widespread clinical application.

Background: Thyroid-associated ophthalmopathy (TAO) is an autoimmune inflammatory disorder, which lacks effective treatment currently. Spica Prunellae (SP) is popularly used for its anti-inflammatory and immune-regulating properties, indicating SP may have potential therapeutic value in TAO. Therefore, the purpose of this study is to identify the efficiency and potential mechanism of SP in treating TAO. Methods: A network pharmacology integrated molecular docking strategy was used to predict the underlying molecular mechanism of treating TAO. Firstly, the active compounds of SP were obtained from TCMSP database and literature research. Then we collected the putative targets of SP and TAO based on multi-sources databases to generate networks. Network topology analysis, GO and KEGG pathway enrichment analysis were performed to screen the key targets and mechanism. Furthermore, molecular docking simulation provided an assessment tool for verifying drug and target binding. Results: Our results showed that 8 targets (PTGS2, MAPK3, AKT1, TNF, MAPK1, CASP3, IL6, MMP9) were recognized as key therapeutic targets with excellent binding affinity after network analysis and molecular docking-based virtual screening. The results of enrichment analysis suggested that the underlying mechanism was mainly focused on the biological processes and pathways associated with immune inflammation, proliferation, and apoptosis. Notably, the key pathway was considered as the PI3K-AKT signaling pathway. Conclusion: In summary, the present study elucidates that SP may suppress inflammation and proliferation and promote apoptosis through the PI3K-AKT pathway, which makes SP a potential treatment against TAO. And this study offers new reference points for future experimental research and provides a scientific basis for more widespread clinical application. [ABSTRACT FROM AUTHOR]

[Objectives] This study aimed to establish HPLC chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae. [ Methods ] The chromatographic conditions were as follows : column, SH1SEIDO CAPCELL PAK CiS MGII column (4.6 mm x250 mm, 5 jxm) ; mobile phase, acetonitrile-0.1% phosphoric acid; gradient elution; detection wavelength, 280 nm; flow rate, 1.0 mL/min; and column temperature, 30 °C. The correlation between the decoction pieces, standard decoction and formula granules of Spica Prunellae was analyzed by specific chromatograms. [Results] The fingerprint chromatograms of decoction pieces, standard decoction and formula granules of Spica Prunellae showed five common peaks, with good correlation. Among the five common peaks, four of them were tanshi- nol, protocatechuic acid, caffeic acid and rosmarinic acid. [Conclusions] The main chemical constituents of decoction pieces, standard decoction and formula granules of Spica Prunellae are basically the same. The HPLC specific chromatogram established can be used for the quality control of Spica Prunellae formula granules. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to explore the molecular mechanisms underlying the effects of Spica prunellae on proliferation and apoptosis of lung cancer cells in vitro.Materials and Methods: The cell viability was determined with the CCK8 assay and the apoptosis was examined by flow cytometry. Western blotting was applied to detect the protein level of caspase-3 expression.Results: It was observed that Spica prunellae could promote the apoptosis and inhibit the proliferation of A549 in vitro. The analysis of Western blotting showed that the expression level of proapoptosis protein caspase-3 was generally unchanged, whereas the level of activated caspase-3 was significantly increased.Conclusion: The results indicated that the growth of lung cancer cells A549 might be inhibited with Spica prunellae through activating the proapoptotic protein caspase-3 and inducing cellular apoptotic pathway. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Spica prunellae (SP) is a well-known traditional Chinese medicinal herb with properties of antihypertensive, antihyperglycemic, antiviral, anti-inflammatory, and antitumor activities. This herb is also popularly consumed as a food additive in some drinks or other food forms for treating pyreticosis. Rosmarinic acid (RA) is the marker compound from SP, which possesses anti-oxidative and anti-inflammatory functions. Aim of the study This study aims to investigate the regulatory effect of the water extract of SP (WESP) and RA on efflux transports (ETs), including P-glycoprotein ( p -gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) in HepG2 cell line. Results would provide beneficial information for the proper application of SP in clinics. Materials and methods HepG2 cells were treated with different doses of the tested drugs for 24 or 96 h. MTT assay was used to examine cell viability. The protein and mRNA levels of the ETs were measured by using Western blot and real-time PCR, respectively. Reporter assay was used to study the antioxidant response element (ARE)-luciferin activity by using HepG2-C8 cells, which were generated by transfecting plasmid containing ARE-luciferin gene into HepG2 cells. The transport activities of ETs were tested by using substrate probes. Results WESP significantly ( p <0.05) increased the expression of ETs in a dose-dependent manner. The increase caused by WESP was stronger than RA alone. Both WESP and RA promoted the translocation of nuclear factor E2-related factor-2 (Nrf2) from cytoplasm to the nucleus as well as significantly ( p <0.05) enhanced the ARE-luciferin activity. WESP and RA also enhanced the efflux activity of P-gp and MRP2, accompanied by marked increase ( p <0.05) in the intracellular ATP levels. Conclusions WESP could significantly induce the expression of ETs through the activation of Nrf2-mediated signaling pathway in HepG2 cells. RA could be one of the active compounds responsible for the induction. WESP and RA also enhanced the efflux activity of P-gp and MRP2, and the increased intracellular ATP levels were likely involved in this induction. Results of this study provide a better understanding of the regulation of SP on ETs and the underlying molecular mechanism. Results indicated that potential drug–drug interactions may exist when SP is co-administered with other substrate drugs that are transported via the ETs, especially P-gp and MRP2, thereby providing beneficial information for appropriate use of SP for clinical therapy. [ABSTRACT FROM AUTHOR]

Spica Prunellae has long been used as a significant component in numerous traditional Chinese medicine (TCM) formulas to clinically treat cancers. Previously, Spica Prunellae was shown to promote cancer cell apoptosis and inhibit angiogenesis in vivo and in vitro. To further elucidate the precise mechanism of its tumoricidal activity, the effect of the ethanol extract of Spica Prunellae (EESP) on the proliferation of human colon carcinoma HT-29 cells was elucidated and the underlying molecular mechanisms were investigated. The proliferation of HT-29 cells was evaluated using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation analyses. The cell cycle was determined using fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The mRNA and protein expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1 was examined using RT-PCR and western blotting, respectively. EESP was observed to inhibit HT-29 viability and survival in a dose- and time-dependent manner. Furthermore, EESP treatment blocked Gj/S cell cycle progression and reduced the expression of pro-proliferative cyclin D1 and CDK4 at the transcriptional and translational levels. Altogether, these data suggest that the inhibition of cell prolif- eration via Gj/S cell cycle arrest may be one of the mechanisms through which Spica Prunellae treats cancer. [ABSTRACT FROM AUTHOR]

Background: Constitutive activation of STAT3 is one of the major oncogenic pathways involved in the development of various types of malignancies including colorectal cancer (CRC); and thus becomes a promising therapeutic target. Spica Prunellae has long been used as an important component in many traditional Chinese medicine formulas to clinically treat CRC. Previously, we found that Spica Prunellae inhibits CRC cell growth through mitochondrion-mediated apoptosis. Furthermore, we demonstrated its anti-angiogenic activities in vivo and in vitro. To further elucidate the precise mechanism of the potential tumoricidal activity of Spica Prunellae, using a CRC mouse xenograft model, in this study we evaluated its therapeutic efficacy against CRC and investigated the underlying molecular mechanisms. Methods: CRC mouse xenograft model was generated by subcutaneous injection of human colon carcinoma HT-29 cells into nude mice. Animals were given intra-gastric administration with 6 g/kg of the ethanol extract of Spica Prunellae (EESP) daily, 5 days a week for 16 days. Body weight and tumor growth were measured every two days. Tumor growth in vivo was determined by measuring the tumor volume and weight. HT-29 cell viability was examined by MTT assay. Cell apoptosis and proliferation in tumors from CRC xenograft mice was evaluated via immunohistochemical staining (IHS) for TUNEL and PCNA, and the intratumoral microvessel density (MVD) was examined by using IHS for the endothelial cell-specific marker CD31. The activation of STAT3 was evaluated by determining its phosphorylation level using IHS. The mRNA and protein expression of Bcl-2, Bax, Cyclin D1, VEGF-A and VEGFR2 was measured by RT-PCR and IHS, respectively. Results: EESP treatment reduced tumor volume and tumor weight but had no effect on body weight change in CRC mice; decreased HT-29 cell viability in a dose-dependent manner, suggesting that EESP displays therapeutic efficacy against colon cancer growth in vivo and in vitro, without apparent toxicity. In addition, EESP significantly inhibited the phosphorylation of STAT3 in tumor tissues, indicating its suppressive action on the activation of STAT3 signaling. Consequently, the inhibitory effect of EESP on STAT3 activation resulted in an increase in the pro-apoptotic Bax/Bcl-2 ratio, decrease in the expression of the pro-proliferative Cyclin D1 and CDK4, as well as down-regulation of pro-angiogenic VEGF-A and VEGFR-2 expression. Finally, these molecular effects led to the induction of apoptosis, the inhibition of cell proliferation and tumor angiogenesis. Conclusions: Spica Prunellae possesses a broad range of anti-cancer activities due to its ability to affect STAT3 pathway, suggesting that Spica Prunellae could be a novel potent therapeutic agent for the treatment of CRC. [ABSTRACT FROM AUTHOR]

Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti-HIV herbal drugs has proceeded slowly, partly because of the lack of a high-throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti-HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C-X-C receptor 4 ( CXCR4) and C-C receptor 5 ( CCR5) promoter activities. A single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV-1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down-regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 µ m) inhibited HIV-1 in a dose-dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers ( p < 0.05). Spica Prunellae and Herba Andrographitis (EC50: 3.18 and 5.49 µg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti-HIV-1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV-1 replication. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The immunosuppressive activity of the ethanol extract of Spica Prunellae (Prunella vulgaris fruiting spikes) (EESP) consisting of a mixture of triterpenoids, flavonoids, tannins and polysaccharide was studied on the immune responses in mice. The effects of EESP on mice splenocyte proliferation in vitro were measured. EESP significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. Furthermore, the effects of EESP at three dose levels (a single dose of 0.25, 0.5, and 1.0mg) on the humoral and cellular immune responses of mice subcutaneously immunized with ovalbumin (OVA) were examined. ICR mice were immunized subcutaneously with OVA on day 0 and 14. Starting on the day of immunization, the mice were administered subcutaneously with EESP at 0.25, 0.5, and 1.0mg at intervals of 7 days for a total of five doses. On day 28, antigen-specific antibodies in serum, mitogen- and OVA-induced splenocyte proliferation were investigated. EESP significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation in the immunized mice in a dose-dependent manner. Total IgG, IgG1 and IgG2b levels in the immunized mice were significantly reduced by EESP. Moreover, the suppressing effects on the antibody responses to OVA in the immunized mice were dose-dependently enhanced according to the increase of EESP. The results suggest that EESP could suppress the cellular and humoral response in mice. [Copyright &y& Elsevier]

Background: As a well-known herb used in the treatment of colon adenocarcinoma (COAD), Spica Prunellae (SP) shows favorable clinical effect and safety in China for many years, but its active ingredients and therapeutic mechanisms against COAD remain poorly understood. Therefore, this study aims to uncover active ingredients and mechanisms of SP in the treatment of COAD using a combined approach of network pharmacology and bioinformatics., Methods: A comprehensive approach mainly comprised of target prediction, network construction, pathway and functional enrichment analysis, and hub genes verification was adopted in the current study., Results: We collected 102 compounds-related genes and 3549 differently expressed genes (DEGs) following treatment with SP, and 64 disease-drug target genes between them were recognized. In addition, a total of 25 active ingredients in SP were identified. Pathway and functional enrichment analyses suggested that the mechanisms of SP against COAD might be to induce apoptosis of colon cancer cells by regulating PI3K-Akt and TNF signaling pathways. Recognition of hub genes and core functional modules was performed by constructing protein-protein interaction (PPI) network, from which TP53, MYC, MAPK8 and CASP3 were found as the hub target genes that might play an important part in therapy for COAD. Subsequently we further compared the differential expression level and assessed the prognostic value of these four hub genes. These result of verification suggested that SP exerted therapeutic effects against COAD via a PPI network involving TP53, MYC, MAPK8 and CASP3., Conclusion: In this study, active ingredients and mechanisms of SP in the treatment of COAD were systematically discussed, which provided the foundation for further experimental studies and might act to promote its appropriate clinical application., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Spica Prunellae has long been used as a significant component in numerous traditional Chinese medicine (TCM) formulas to clinically treat cancers. Previously, Spica Prunellae was shown to promote cancer cell apoptosis and inhibit angiogenesis in vivo and in vitro . To further elucidate the precise mechanism of its tumoricidal activity, the effect of the ethanol extract of Spica Prunellae (EESP) on the proliferation of human colon carcinoma HT-29 cells was elucidated and the underlying molecular mechanisms were investigated. The proliferation of HT-29 cells was evaluated using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation analyses. The cell cycle was determined using fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The mRNA and protein expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1 was examined using RT-PCR and western blotting, respectively. EESP was observed to inhibit HT-29 viability and survival in a dose- and time-dependent manner. Furthermore, EESP treatment blocked G 1 /S cell cycle progression and reduced the expression of pro-proliferative cyclin D1 and CDK4 at the transcriptional and translational levels. Altogether, these data suggest that the inhibition of cell proliferation via G 1 /S cell cycle arrest may be one of the mechanisms through which Spica Prunellae treats cancer.

The objective of the study was to investigate the in-vivo anti-tumour activity of Spica prunellae extract, and to preliminarily explore the possible mechanism of in-vivo anti-tumour effect of Spica prunellae extract. Tumour inhibition rate and tumour apoptosis-related protein status were determined using the mice model of transplanted T lymphoma cell EL-4 tumour, and by immunohistochemical method. The results revealed that Spica prunellae extract showed certain tumour inhibitory effect, and compared with the model group. Tumour weight in Spica prunellae high-dose group was highly significantly different (P<0.01). Tumour weight in Spica prunellae medium-dose group was also significantly different (P<0.05) compared with the model group. Spica prunellae high-dose group enabled the high expression of Bcl-2 protein (47.54%) and low expression of Bax protein (13.14%). The study concluded that Spica prunellae extract has certain in vivo anti-tumour effect, which may be achieved through regulation of apoptosis related proteins.