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A polysaccharide fraction from medicinal herb Prunella vulgaris downregulates the expression of herpes simplex virus antigen in Vero cells.

Herpes simplex viruses (HSV) are pathogenic. With the emergence of drug-resistant strains of HSV, new antiviral agents, especially those with different modes of action, are urgently needed. Prunella vulgaris L. (Labiatae), a perennial plant commonly found in China and Europe, has long been used as a folk medicine to cure ailments. In this study, a polysaccharide fraction was prepared from Prunella vulgaris (PPV), and its effects on the expressions of HSV-1 and HSV-2 antigens in their host Vero cells were investigated with flow cytometry. The HSV antigen increased time-dependently in the infected cells, and PPV reduced its expression. The effective concentrations of PPV with 50% reductions of the HSV-1 and HSV-2 antigens were 20.6 and 20.1 microg/ml, respectively. The novelty of PPV is that it also reduces the antigen expression of acyclovir-resistant strain of HSV-1. After incubations with 25-100 microg/ml of PPV the HSV antigen-positive cells were reduced by 24.8-92.6%, respectively, showing that this polysaccharide fraction has a different mode of anti-HSV action from acyclovir. Results from this study show that PPV is effective against both the HSV-1 and HSV-2 infections, and flow cytometry offers a quantitative and highly reproducible anti-HSV drug-susceptibility assay.

Animals↗

In vitro and in vivo immunosuppressive activity of Spica Prunellae ethanol extract on the immune responses in mice.

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 1.0 mg) 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 1.0 mg 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.

Animals↗

Isolation and characterization of an anti-HSV polysaccharide from Prunella vulgaris.

A water soluble substance was isolated from a Chinese herb, Prunella vulgaris, by hot water extraction, ethanol precipitation and gel permeation column chromatography. Chemical tests showed that the substance was an anionic polysaccharide. Using a plaque reduction assay, the polysaccharide at 100 microg/ml was active against the herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), but was inactive against cytomegalovirus, the human influenza virus types A and B, the poliovirus type 1 or the vesicular stomatitis virus. The 50% plaque reduction dose of the polysaccharide for HSV-1 and HSV-2 was 10 microg/ml. Clinical isolates and known acyclovir-resistant (TK-deficient or polymerase-defective) strains of HSV-1 and HSV-2 were similarly inhibited by the polysaccharide. Pre-incubation of HSV-1 with the polysaccharide at 4, 25 or 37 degrees C completely abrogated the infectivity of HSV-1, but pre-treatment of Vero cells with the polysaccharide did not protect cells from infection by the virus. The addition of the polysaccharide at 0, 2, 5.5 and 8 h post-infection of Vero cells with HSV-1 at a multiplicity of infection (MOI) of five reduced the 20 h-yield of intracellular infectious virus by 100, 99, 99 and 94%, respectively. In contrast, a similar addition of heparin showed 85, 63, 53 and 3% reduction of intracellular virus yield, respectively. These results suggest that the polysaccharide may inhibit HSV by competing for cell receptors as well as by some unknown mechanisms after the virus has penetrated the cells. The Prunella polysaccharide was not cytotoxic to mammalian cells up to the highest concentration tested, 0.5 mg/ml and did not show any anti-coagulant activity. In conclusion, the polysaccharide isolated from P. vulgaris has specific activity against HSV and its mode of action appears to be different from other anionic carbohydrates, such as heparin.

Animals↗

Biological activities of Prunella vulgaris extract.

The organic fraction (OF; 25.7% w/w of rosmarinic acid) of Prunella vulgaris (total extract) was found to exhibit the following: scavenging activity on diphenylpicrylhydrazyl radical (DPPH), inhibition of in vitro human LDL Cu(II)-mediated oxidation, protection of rat mitochondria and rat hepatocytes exposed to either tert-butyl hydroperoxide, or to Cu(II) and Fe(III) ions. OF also showed a potential to inhibit rat erythrocyte haemolysis and it reduced the production of LTB(4) in bovine PMNL generated by the 5-lipoxygenase pathway. Other observations included antiproliferative effects against HaCaT cells and mouse epidermal fibroblasts and a moderate OF antimicrobial activity on gram-positive bacteria. Rosmarinic, caffeic and 3-(3,4-dihydroxyphenyl)lactic acids exhibited less potent activity than the plant extract in all bioassays. The antioxidative, antimicrobial, together with antiviral effects offer good prospects for the medicinal applications of P. vulgaris.

Animals↗

Cytoprotectivity of Prunella vulgaris on doxorubicin-treated rat cardiomyocytes.

The cardioprotective effect of Prunella vulgaris ethylacetate fraction (PVEF) and its constituent rosmarinic acid (RA) was evaluated on isolated rat cardiomyocytes subjected to doxorubicin-induced oxidative stress. The cytoprotectivities of PVEF and RA were concentration-dependent in the range of 0.005 to 0.05 mg/ml and the effect of PVEF correlated with the RA content. Dexrazoxan (DE), used as positive control, was less effective than PVEF or RA.

Animals↗

Phenolics-rich extracts from Silybum marianum and Prunella vulgaris reduce a high-sucrose diet induced oxidative stress in hereditary hypertriglyceridemic rats.

The study tested the effects of phenolics-rich extracts from the plants Silybum marianum (silymarin) and Prunella vulgaris (PVE) on blood and liver antioxidant status and lipoprotein metabolism. Hereditary hypertriglyceridemic rats fed on standard diet (STD) or high-sucrose diet (HSD, 70cal% of sucrose) for two weeks were used. HSD doubled plasma and liver triacylglycerol (TAG) and increased plasma VLDL-TAG and VLDL-cholesterol compared to STD. Administration of silymarin or PVE as 1% dietary supplements in HSD did not influence lipid levels in plasma or liver, but both extracts caused decrease in plasma VLDL-cholesterol levels. HSD-induced oxidative stress was manifested in increased TBARS and conjugated dienes (CD) content, decreased GSH levels and glutathione peroxidase (GPX) activity in blood and liver. In blood the activity of superoxide dismutase (SOD) decreased, whereas in liver the activity of catalase increased after HSD. Feeding on HSD containing phenolics-rich extracts resulted in reduction of TBARS and CD content and in increase of blood GPX activity and elevated GSH content in liver. Besides, silymarin increased the activity of SOD and level of GSH in blood. Catalase activity in blood or liver was not influenced by the presence of plant extracts in the diet. These results indicate that silymarin and PVE improve antioxidant status in blood and liver and positively affect plasma lipoprotein profile in an experimental model of dietary induced hypertriglyceridemia.

Animals↗

[Determination of oleanolic acid and ursolic acid in spica Prunellae by derivative GC method].

OBJECTIVE: To develop a GC method to determine the oleanolic acid and ursolic acid in Spica Prunellae. METHOD: Before GC analysis, the sample was derivatized with CH2N2 solution. The GC conditions were as follows: comumn-10% SE-30(2m x 3mm) and column temperature -270 degrees C. RESULT: The two constituents were well separated and had good linearity in the range of 0.0025-0.4000 mg/ml. The average recoveries and RSD of oleanolic acid were 93.53% and 3.5%, 94.18% and 3.0% respectively. CONCLUSION: The method is good for determining oleanolic acid and ursolic acid in Chinese medicines.

Chromatography, Gas↗

[Triterpenoid compounds of Prunella genus and their features of 13C NMR spectroscopy].

OBJECTIVE: To summarize the research advances and 13C NMR features of triterpenoid compounds in the plant of Prunella genus. METHODS: Consulting related literatures and summing up the triterpenoid compounds and their features of 13C NMR spectra. RESULT AND CONCLUSION: Up to now, 28 triterpenoid compounds were isolated from the plant of P. genus. of which 20 were in free state and 8 were triterpenoid saponins. The 13C NMR spectra of different triterpenoids showed different features in respects such as skeletons, substituent groups and positions.

Magnetic Resonance Spectroscopy↗

Immune modulatory effects of Prunella vulgaris L.

Prunella vulgaris L. (Labiatae) is a perennial plant known as 'self-heal' in Western herbal medicine. It has a wide array of biological effects exhibiting numerous therapeutic potentials. Its anti-microbial effects including anti-viral and anti-bacterial effects are, presently, receiving increasing attention. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects or actions remain unknown. In view of the fact that polysaccharides isolated from medicinal herbs often function as biological response modifier of body immunity, we hypothesized that the anti-microbial effect of polysaccharides isolated from P. vulgaris is probably also mediated via immune modulation. We have isolated four polysaccharides containing fractions from P. vulgaris, one of the fractions, PV2, could markedly stimulate the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2 stimulation was as high as that seen in stimulation using bacterial endotoxin lipopolysaccharide (LPS), and this stimulatory response is dose-dependent. In addition to monocyte/macrophage, PV2 also stimulated the proliferation of splenocytes. In this study, we have shown that the polysaccharides isolated from P. vulgaris have marked immune stimulatory effects, which may bring about the anti-microbial effects of P. vulgaris.

Animals↗

Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris.

Crude extracts of four Chinese herbs, Arctium lappa, Astragalus membranaceus, Andrographis paniculata, and Prunella vulgaris, were assessed in several tissue culture lines for anti-HIV activity and for cytotoxicity. One extract, obtained from P. vulgaris, was able to significantly inhibit HIV-1 replication with relatively low cytotoxicity. The active factor was purified using sequential precipitations with ethanol and n-butanol, followed by reverse-phase and gel permeation high-performance liquid chromatographic separations. The active component was anionic with a molecular weight of approximately 10 kDa. The purified extract inhibited HIV-1 replication in the lymphoid cell line MT-4, in the monocytoid cell line U937, and in peripheral blood mononuclear cells at effective concentrations of 6, 30, and 12.5 micrograms/ml, respectively. Pretreatment of uninfected cells with the extract prior to viral exposure did not prevent HIV-1 infection. By contrast, preincubation of HIV-1 with the purified extract dramatically decreased infectiousness. The purified extract was also able to block cell-to-cell transmission of HIV-1, prevented syncytium formation, and interfered with the ability of both HIV-1 and purified gp120 to bind to CD4. PCR analysis confirmed the absence of HIV-1 proviral DNA in cells exposed to virus in the presence of the extract. These results suggest that the purified extract antagonizes HIV-1 infection of susceptible cells by preventing viral attachment to the CD4 receptor.

Astragalus propinquus↗

Isolation, purification, and partial characterization of prunellin, an anti-HIV component from aqueous extracts of Prunella vulgaris.

Prunellin, an anti-HIV active compound, was isolated from aqueous extracts of the Chinese medicinal herb, Prunella vulgaris, and purified to chromatographic homogeneity. Infrared and NMR spectroscopy identified prunellin as a polysaccharide. Elemental analyses, precipitation with calcium(II), barium(II), or 9-aminoacridine suggest a sulfated polysaccharide. Paper chromatography of the exhaustively hydrolyzed material indicates the presence of glucose, galactose, xylose, gluconic acid, galactonic acid and galactosamine as the constituent monosaccharides. The molecular size of prunellin, as determined by gel permeation chromatography and the Squire method on Sephadex G-75, is about 10 kDa.

Antiviral Agents↗

Identification of inhibitors of the HIV-1 gp41 six-helix bundle formation from extracts of Chinese medicinal herbs Prunella vulgaris and Rhizoma cibotte.

An increasing portion of patients with HIV infection and/or AIDS cannot use currently FDA-approved anti-HIV drugs, including the reverse transcriptase and protease inhibitors, due to the adverse effects and the emergence of drug resistance. Thus, it is essential to develop new anti-HIV agents with a target different from the HIV reverse transcriptase and protease. Using a conformation-specific monoclonal antibody NC-1, we previously established a high throughput screening assay for identification of small molecular organic compounds that disrupt the HIV-1 gp41 six-helix bundle formation, a critical step of membrane fusion between the HIV and the target cell. In the present study, we used this assay to screen for inhibitors of the gp41 six-helix bundle formation from aqueous extracts of nine Chinese medicinal herbs with antiviral activity. We found that the extracts of two herbs, Prunella vulgaris and Rhizoma cibotte, showed potent inhibitory activity. The inhibitory activity of these two herb extracts significantly decreased after they were passed through polyamide resin mini-columns, which are able to bind polyphenols including tannin, an HIV-1 inhibitor with multiple mechanisms of action. The bound polyphenols were eluted from the polyamide columns and also showed potent inhibitory activity on the gp41 six-helix bundle formation. Tannin purchased from different commercial sources inhibited the gp41 six-helix bundle formation in a manner similar to the polyphenols isolated from the herb extracts. These results suggest that tannin may be one of major inhibitors of the HIV-1 gp41 six-helix bundle formation in the herb extracts and that tannin may inhibit HIV-1 entry by disrupting the gp41 six-helix bundle formation.

Antibody Specificity↗

Anti-allergic and anti-inflammatory triterpenes from the herb of Prunella vulgaris.

The activity-guided fractionation of the extract of the herb of Prunella vulgaris (Labiatae) led to the isolation of four triterpenes, i.e., betulinic acid, ursolic acid, 2 alpha,3 alpha-dihydroxyurs-12-en-28-oic acid, and 2 alpha-hydroxyursolic acid. One of these compounds, 2 alpha,3 alpha-dihydroxyursolic acid, demonstrated significant inhibition on the release of beta-hexosaminidase from the cultured RBL-2H3 cells in a dose-dependent manner; the IC50 value was calculated to be 57 microM. When the isolated compounds were tested for their effects on the production of nitric oxide from cultured murine macrophages, RAW 264.7 cells, ursolic acid and 2 alpha-hydroxyursolic acid exhibited strong inhibitory activities (IC50 values, 17 and 27 microM, respectively).

Animals↗

Inhibition of immediate-type allergic reactions by Prunella vulgaris in a murine model.

We studied the effect of aqueous extract of Prunella vulgaris (Labiatae) (PVAE) on immediate-type allergic reactions. PVAE (0.005 to 1 g/kg) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/ 80 in rats. When PVAE was given as pretreatment, at concentrations ranging from 0.005 to 1 g/kg, the serum histamine levels induced by compound 48/ 80 were reduced in a dose-dependent manner. PVAE (0.001 to 1 g/kg) inhibited the passive cutaneous anaphylaxis activated by anti-dinitrophenyl (DNP) IgE antibody dose dependently. PVAE also inhibited the histamine release induced by compound 48/80 or anti-DNP IgE from the rat peritoneal mast cells (RPMC). The level of cyclic AMP in RPMC, when PVAE was added, significantly increased, compared with that of normal control. Moreover, PVAE (0.01 and 0.1 mg/ml) had a significant inhibitory effect on anti-DNP IgE-mediated tumor necrosis factor-alpha production from RPMC. These results indicate that PVAE inhibits immediate-type allergic reactions in rats.

Anaphylaxis↗

Extract of Prunella vulgaris spikes inhibits HIV replication at reverse transcription in vitro and can be absorbed from intestine in vivo.

It has been reported that extracts of the spike of Prunella vulgaris (PS) exhibit anti-HIV activity at the adsorption and reverse transcription stages. In this study, the actual activity of PS in cells, kinetic analysis of the inhibitory activity of PS against HIV reverse transcriptase and the feasibility of oral administration were examined. First, to clarify whether this extract shows anti-HIV activity in cells in vitro, the number of copies of proviral DNA in HIV-exposed cells was calculated. The number of copies was significantly decreased in cells cultured in the presence of PS extract, but not in the presence of dextran sulphate. The activity of PS extract in the cells was also assessed by the drug addition test, during and after HIV adsorption. PS extract and dextran sulphate suppressed HIV production to similar levels when added after HIV adsorption. However, only PS extract suppressed HIV production at the same concentration when the drugs were added during HIV adsorption. Presumably, the penetration of the PS extract into the cells was required for this activity. Secondly, fractionated PS inhibited HIV reverse transcription in a non-competitive manner. This fractionated PS kept anti-HIV activity, but inhibited HIV replication and adsorption to a lesser extent compared to dextran sulphate. Lastly, an active component(s) was detected in plasma in vivo, after injection into the intestine, which demonstrates the feasibility of oral administration dosing.

Animals↗

Is the Prunella (Lamiaceae) hybrid zone structured by an environmental gradient? Evidence from a reciprocaltransplant experiment.

Hybrid zones may be structured by environmentally independent selection against intrinsically unfit hybrids (tension zone models), or by environmentally dependent fitness differences among parental species and hybrids (ecological selection-gradient models). A 30-m slope in a mountain grassland harbors a hybrid zone of the clonal perennials, Prunella grandiflora and P. vulgaris (Lamiaceae), with P. grandiflora in the upper, P. vulgaris in the lower, and both parental species and P. grandiflora × P. vulgaris Hybrids in a narrow middle part. We found gradients for soil depth and water content, and vegetation height and biomass along the slope. A reciprocal transplant experiment yielded crossing reaction norms for vegetative reproduction. Parental species were locally adapted to their home sites, while the three taxa did not differ in vegetative reproduction in the Hybrid transplant site. Local adaptation for vegetative reproduction of P. grandiflora was mediated through higher survival and that of P. vulgaris through higher ramet number, indicating adaptation of their clonal growth strategies (phalanx vs. guerrilla) to the different habitats. Hybrid performance was intermediate between that of the parental species in all three sites, although Hybrids flowered more often than the parental species in the Hybrid site. Our results support ecological selection-gradient rather than tension zone models.

Journal Article↗

[Prunella vulgaris L.--a rediscovered medicinal plant].

In the past, the self-heal (Prunella vulgaris L.) was primarily used as a remedy alleviating pains in the throat, fevers and accelerating wound healing. A high content of rosmarinic acid, immunomodulation effects of the polysaccharide prunelline and antiviral activity of some constituents make the plant interesting from the viewpoint of therapeutical applications. The paper summarizes the contemporary phytochemical knowledge about the self-heal and the results of pharmacological studies of the extracts and pure substances from this plant.

Plant Extracts↗

Macleya cordata and Prunella vulgaris in oral hygiene products - their efficacy in the control of gingivitis.

A double-blind, placebo-controlled clinical trial was performed to investigate the effectiveness of a herbal-based dentifrice in the control of gingivitis. Forty volunteers completed the 84-day study. All subjects were balanced for parameters measured - plaque index (PI), community periodontal index of treatment needs (CPITN) and papillary bleeding index (PBI). The dentifrice was effective in reducing symptoms of gingivitis as evaluated by the CPITN and PBI indexes.

Adult↗