Prunella vulgaris has been used therapeutically for inflammation-related conditions for centuries, but systematic studies of its anti-inflammatory activity are lacking and no specific active components have been identified. In this study, water and ethanol extracts of four P. vulgaris accessions were applied to RAW 264.7 mouse macrophages, and the ethanol extracts significantly inhibited lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production at 30 μg/mL without affecting cell viability. Extracts from different accessions of P. vulgaris were screened for anti-inflammatory activity to identify accessions with the greatest activity. The inhibition of PGE2 and NO production by selected extracts was dose-dependent, with significant effects seen at concentrations as low as 10 μg/mL. Fractionation of ethanol extracts from the active accession, Ames 27664, suggested fractions 3 and 5 as possible major contributors to the overall activity. Rosmarinic acid (RA) content inP. vulgaris was found to independently inhibit inflammatory response, but it only partially explained the extracts' activity. LPS-induced cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) protein expression were both attenuated by P. vulgaris ethanol extracts, whereas RA inhibited only COX-2 expression. Prunella vulgaris has been used therapeutically for inflammation-related conditions for centuries, but systematic studies of its anti-inflammatory activity are lacking and no specific active components have been identified. In this study, water and ethanol extracts of four P. vulgaris accessions were applied to RAW 264.7 mouse macrophages, and the ethanol extracts significantly inhibited lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production at 30 μg/mL without affecting cell viability. Extracts from different accessions of P. vulgaris were screened for anti-inflammatory activity to identify accessions with the greatest activity. The inhibition of PGE2 and NO production by selected extracts was dose-dependent, with significant effects seen at concentrations as low as 10 μg/mL. Fractionation of ethanol extracts from the active accession, Ames 27664, suggested fractions 3 and 5 as possible major contributors to the overall activity. Rosmarinic acid (RA) content in P. vulgaris was found to independently inhibit inflammatory response, but it only partially explained the extracts' activity. LPS-induced cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) protein expression were both attenuated by P. vulgaris ethanol extracts, whereas RA inhibited only COX-2 expression.

Due to the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immune modulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE2) production. HPLC fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of E. purpurea, E. angustifolia, E. pallida, and E. tennesseensis identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharide induced PGE2 production in RAW264.7 mouse macrophage cells. Nitric oxide (NO) production and parallel cytotoxicity screens were also employed to substantiate an anti-inflammatory response. Echinacea pallida showed significant inhibition of PGE2 with a first round fraction, containing GC-MS peaks for Bauer Ketones 20, 21, 22, 23, and 24, with 23 and 24 identified as significant contributors to this PGE2 inhibition. Chemically synthesized Bauer Ketones 21 and 23 at 1 μM each significantly inhibited both PGE2 and NO production. Three rounds of fractionation were produced from an E. angustifolia extract. GC-MS analysis identified the presence of Bauer Ketone 23 in third round Fraction 3D32 and Bauer Alkylamide 11 making up 96% of third round Fraction 3E40. Synthetic Bauer Ketone 23 inhibited PGE2 production to 83 % of control and synthetic Bauer Alkylamide 11 significantly inhibited PGE2 and NO production at the endogenous concentrations determined to be present in their respective fraction, thus each constituent partially explained the in vitro anti-inflammatory activity of their respective fraction. From this study two key contributors to the anti-inflammatory properties of E. angustifolia were identified as Bauer Alkylamide 11 and Bauer Ketone 23.

Hypericum perforatum extracts have been used to treat diseases including mild-to-moderate depression and inflammatory conditions. It is particularly important to identify which constituents present in the H. perforatum extracts are responsible for its' anti-inflammatory activity since consumers are taking H. perforatum preparations to treat inflammation. We used a combination of four putative bioactive constituents, called the 4-component-system, that interacted synergistically to explain the light-activated anti-inflammatory activity of an H. perforatum fraction in RAW 264.7 mouse macrophages and combined the constituents at concentrations detected in the fraction to identify key molecular targets. LPS was used to model an inflammatory response and the 4-component-system and H. perforatum fraction were used as treatments that inhibited LPS induced prostaglandin E 2 (PGE 2 ) production in RAW 264.7 mouse macrophages in studies of gene expression profiles. We used Affymetrix genechips, statistical analysis, and quantitative real-time PCR to identify key gene targets of the 4-component-system and the sub-fraction from an H. perforatum ethanol extract. The H. perforatum sub-fraction with or without LPS stimulation affected far more genes than the 4-component-system with and without LPS. Genes involved in Janus kinase and signal transducer and activator of transcription (JAK-STAT) and eicosanoid pathways were identified that could account for the reduction in PGE 2 seen with both treatments in the LPS-stimulated macrophages. Ten genes may be particularly important targets for the activity of the 4-component-system and the fraction with LPS stimulation and these genes were involved in inflammatory signaling pathways, namely JAK-STAT and eicosanoid pathways. Keywords gene expression; Hypericum perforatum; interactions; RAW 264.7 mouse macrophages; pseudohypericin; amentoflavone; quercetin; chlorogenic acid *To whom correspondence should be addressed: Diane F. Birt, 220 MacKay Hall, Iowa State University; Ames, Iowa, 50011; USA, dbirt@iastate.edu, Fax: (515) 294-6193, Telephone: (515) 294-9873. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptPhytochemistry. Author manuscript; available in PMC 2011 May 1. IntroductionMacrophages can release inflammatory mediators such as prostaglandins and cytokines in response to lipopolysaccharide (LPS) stimulation validating the use of LPS treated macrophages as a model of inflammation. Genes with prominent roles in LPS-induced inflammation include cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF...

An in vitro semi-continuous long-term (3 wk) anaerobic incubation system simulating lower gut fermentation was used to determine variability in gut microbial metabolism between 4 predigested high amylose-resistant starch residues (SR): SRV, SRVI, SRVII, and SRGEMS in human fecal samples. Subjects participated twice, 5 mo apart: 30 in Phase I (15 lean, 9 overweight and 6 obese), 29 in Phase II (15 lean, 9 overweight, 5 obese); 13 of 15 lean subjects participated in both phases. Of the 4 SRs, SRV displayed the highest gelatinization temperature, peak temperature, enthalpy changes, and the least digestibility compared with the other SRs. In both phases, compared with blank controls, all SRs increased butyrate ∼2-fold which stabilized at week 2 and only SRV caused greater propionate concentration (∼30%) after 3 wk which might have been partly mediated by its lesser digestibility. Fecal samples from lean and overweight/obese subjects incubated with SRs showed similar short-chain fatty acid production across both time points, which suggests that resistant starch may benefit individuals across BMIs.

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