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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 RAW264.7 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 cyclooxyginase-2 (COX-2) and nitric oxide synthase (iNOS) protein expression were both attenuated by P. vulgaris ethanol extracts, while RA only inhibited COX-2 expression.
Prunella vulgaris (Lamiaceae), commonly called self-heal, is a perennial herb widely distributed in Asia and Europe. In Europe, P. vulgaris has been a popular traditional remedy since the 17th century for several medical conditions including mild fever, sore throat, and external wound healing (1). In China, P. vulgaris is called ‘Xia Ku Cao’ and has a long history in therapeutic use as an antipyretic, and more recently for anti-keratitis purposes (2). In South Korea, P. vulgaris is applied to patients with goiter, dermatitis, and skin allergy (3). Despite its wide use for health purposes, only a few scientific studies have addressed the health promoting claims of P. vulgaris. Aqueous extracts of P. vulgaris, rich in carbohydrates, were reported to have anti-carcinogenic, counter-UV damage, immune-regulatory, and anti-viral effects (4-6). In addition, increasing evidence suggests that extracts prepared with organic solvents, such as ethanol and methanol, possess anti-estrogenic, anti-inflammatory, and anti-oxidative properties (7-9).
Although most of the bioactivities seen in P. vulgaris water extracts are attributed to polysaccharide compounds, no specific component in these extracts has been associated with anti-inflammatory activity (6). On the other hand, polyphenol-rich aqueous-ethanolic extract (30% v/v) contains two known constituents with anti-inflammatory activity, namely rosmarinic acid (RA) and ursolic acid (UA) (8, 10). Information regarding the relative abundance of possible anti-inflammatory compounds in different populations of P. vulgaris is not available, and direct comparison of activity among different accessions has not been reported.
Inflammation is critical in recruiting immune cells and molecules to the site of infection for defense. However, various kinds of tissue damage and pathological consequences ensue when ‘sterile inflammation’, such as obesity and self-immune diseases, occurs, or infection-induced inflammation becomes chronic. Among participating cells, macrophages play a central role in organizing the release of inflammation mediators, including prostaglandin E2 (PGE2), nitric oxide (NO), and cytokines that promote host protection, as well as causing pathological consequences such as tissue edema and abnormal histological change (8, 11). The RAW 264.7 mouse macrophage cell line is widely used for studies of inflammation, due to its reproducible response to lipopolysaccharide (LPS), mediated by toll-like receptor 4 (TLR4) (12). Our previous research on Hypericum and Echinacea species successfully employed this model to study effects of these botanical materials on cyclooxygenase-2 (COX-2) regulated PGE2 production and upstream intracellular events (13, 14). Other research with these cells indicate a well-defined nitric oxide induction by LPS through inducible nitric oxide synthase (iNOS) (15). Therefore, we used this model to investigate the effect of P. vulgaris extracts on these two major inflammatory mediators.
The main purpose of the current study was to compare anti-inflammatory activity of water and ethanol extracts of P. vulgaris in LPS-stimulated RAW 264.7 mouse macrophages and to assess any differences in activity among different P. vulgaris accessions. Fractionation of active extracts allowed us to explore possible bioactive compounds. At the same time, parallel comparison of pure compounds and extract activities revealed the contribution of selected compounds.
Vegetative samples of P. vulgaris were acquired from the North Central Regional Plant Introduction Station (NCRPIS) of the U.S. Department of Agriculture-Agricultural Research Service (USDA/ARS), Ames, IA. This study included accessions Ames 27664, 27665, and 27666 from North Carolina, Ames 27748 and 28436 (PI 656842) from Missouri, and Ames 28353 (PI 656839), 28354 (PI 656840), 28355, 28356, 28357 (PI 656841), 28358, and 28359 from Iowa. Further detailed information about the origins of these accessions is available from the Germplasm Resources Information Network (GRIN) database at: http://www.ars-grin.gov/npgs/acc/acc_queries.html.
Seeds from original samples of all these accessions were germinated at 25°C in Petri plates and then transferred to greenhouse flats held at 20-25°C. In April 2006, seedlings of Ames 27664, 27665, 27666, and 27748 were transplanted to the field at two months of age, and each individual accession was isolated within control-pollination cages. In October 2007, above-ground portions of these accessions were harvested and air dried at ~40°C for one week. Dried samples were then ground in a Wiley Mill and stored at -20°C under nitrogen until extraction.
In May 2007, two-month-old seedlings of the remaining accessions were transplanted to the same field with the same isolation procedure. In July, 2008, all accessions in the field, except for Ames 27666, were harvested, air-dried, ground and stored, as noted above.
The taxonomic identity of each accession was confirmed at the time of flowering. Seed samples for each accession are conserved and distributed by the NCRPIS, and corresponding voucher specimens are held at the Ada Hayden Herbarium, Iowa State University.
All glassware was heated at 200°C for 2 hours to destroy endotoxin, while other supplies were purchased sterile. Random samples of supernatant and cell pellet were chosen from cell culture in selected experiments for mycoplasma screening with a MycoProbe™ mycoplasma detection kit (R&D Systems, Minneapolis, MN), and no contamination was found. Water, ethanol, and DMSO solvents, along with extraction and fractionation products, were tested for endotoxin by using a Lymulus Amebocyte Lysate Test (Bio Whittaker, Walkersville, MD) (14). The endotoxin levels ranged from undetectable to 0.000658 EU/mL for all extracts and ethanol fractions, well below the 5 EU/mL threshold for significant stimulation of RAW 264.7 macrophages (13). Endotoxin levels of water fractions, ranging from 0.0001 to 0.0044 EU/mL, were higher than those of extracts and ethanol fractions.
Rosmarinic acid and ursolic acid at 90-100% and 95% purity, respectively, (as graded by the manufacturer, Fisher Scientific, Hanover Park, IL) were dissolved in dimethyl sulfoxide (DMSO) to 100 mM and 50 mM stock concentration, respectively, and stored at -20°C.
Ethanol Extraction: Six grams of dried P. vulgaris ground sample was extracted with 500 mL 95% ethanol via Soxhlet for 6 hours. The extract was filtered, then dried by rotary evaporation at <40°C followed by lyophilization. The extract was then dissolved in the minimal amount of DMSO that completely dissolved the residue and stored at -20 °C.
Water Extraction: Six grams of dried P. vulgaris ground sample was extracted with 100 mL boiling endotoxin-free water. The plant material was steeped with stirring for 1 hour, and then filtered through a G6 glass fiber circle (Fisher Scientific, Hanover Park, IL) in a Buchner funnel. The filtrate was centrifuged at 10,000× g for 20 minutes to remove additional particulates. Then, the extract was lyophilized, weighed, and re-dissolved with minimal volume of endotoxin-free water that dissolved the residue and stored at -20 °C.
Size-exclusion chromatography fractionation of the water extract: two grams of dry P. vulgaris Ames 27664 water extract residue, dissolved in 10 ml endotoxin-free water, was loaded onto a 2.5×75 cm Sephacryl 100HR column. Elution was with endotoxin-free water and the eluent was collected in 10 mL individual fractions over the following 72 hours until a combined volume of 2 L was recovered. The absorbance at 210 nm was measured for all tubes. Nine peaks were reserved, of these the last two peaks were pooled in the same fraction due to their low yield resulting in eight fractions that were concentrated by lyophilization. The residues after lyophilization were then completely dissolved in the same volume of water and all fractions were stored at -20 °C.
Semi-preparative HPLC fractionation of the ethanol extract: One hundred milligrams of dry P. vulgaris Ames 27664 ethanol extract residue, dissolved in 0.5 mL 60% ethanol, were loaded onto an YMC-pack ODS-AM 250×10 mm C18 column (YMC, Kyoto, Japan). The HPLC system used was a Beckman-Coulter System Gold with a 126 solvent module, a Model 508 autosampler and a Model 168 detector. Solvents were endotoxin-free water containing 0.1% acetic acid as A and acetonitrile as B, following the gradient shown in Table 1. Based on absorbance peaks at 210 nm, 2 mL aliquots of fractions from the ethanol extract were pooled into seven fractions, which were concentrated by lyophilization and later dissolved with same amount of DMSO for storage at -20 °C.
RAW 264.7 mouse macrophage cells (American Type Culture Collection, Manassas, VA) were cultured as described by Hammer et al. (13) in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 10% sodium bicarbonate and 100 IU/mL penicillin/streptomycin (all from Invitrogen, Carlsbad, CA). The cells were incubated at 37 °C, 5% CO2, and grew to 80% confluency before passaging or plating. Cells were plated in 24, 48-well-plates, or on Petri dishes (Corning, Lowell, MA) for treatment as described by Hammer et al. (13). When the plated cells reached 80% (for 8 hour treatment) or 60% (for 24 hour treatment) confluency, the stock of P. vulgaris extracts and fractions, or pure compounds was diluted to 1000× treatment concentration with vehicle solvent and then added to media at 0.1%. Treatments in 500 μL media were applied to macrophages in 24 well plates for PGE2 and NO assay. For cytotoxicity assay, 300 μL of treatment was applied to the 48 well plates. For Western blotting, treatments in 10 mL media were added to the Petri dishes. Except for the cytotoxicity assay which was conducted without induction, each treatment was administrated with and without stimulation by 1 μg/mL LPS (Escherichia coli 02B:B6) (Sigma, St. Louis, MO). Three replicates were included on three individual plates. Aside from media control and DMSO vehicle control at 0.1% v/v, a 10 μM (100 μM for Western blotting) quercetin (Sigma, St. Louis, MO) treatment was also included as a positive control in each experiment to validate each assay because of its proven anti-inflammatory activity as well as its presence in P. vulgaris ethanol extract at a trace amount (16). Two additional strategies were also attempted for PGE2 assay, one using a 30-minute pre-treatment with extracts before LPS induction, and the other using a 2-hour LPS induction before administration of plant extracts.
Cytotoxicity was assayed on all extracts, fractions, and pure compounds following a protocol modified from Schmitt et al. (17). Ursolic acid, a known cytotoxic constituent of P. vulgaris plant material that accounts for 0.05-0.2% dry matter (18), was used as the positive control at 10, and 30 μM. The maximal treatment dose used in the bio-activity assays was applied to cells for 24 hours in 48-well plates before being replaced by 200 μL fresh media and 30 μL of Celltiter96 Aqueous One Solution Cell Proliferation Assay solution (Promega, Madison, WI). After an 195-minute incubation, 200 μL of metabolized dye product was transferred from each well to a 96 well plate and absorbance was read by a plate reader at 562 nm. Percentages of viability compared to media+DMSO control were determined for all treatments.
Treatment lasted 8 hours for PGE2 assay, and 24 hours for NO assay. before all supernatant in each well was collected on ice for future assays (19). After collection, the supernatant was kept at -80 °C before the PGE2 assay was conducted. Samples were diluted (1:15) and analyzed with Biotrek™ PGE2 enzyme immune assay (EIA) (GE healthcare, Piscataway, NJ) according to manufacturer's protocol. PGE2 concentrations in the supernatant samples were determined by comparison with a standard curve.
For the NO assay, the supernatant was collected after 24-hour treatment and stored at 4 °C before analysis. Griess reagent (Promega, Madison, WI) was employed to indirectly measure NO production in cell culture by measuring nitrite concentration (20). Fifty micro-liters of supernatant sample or a series of standards were mixed with 50 μL of sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) in a 96-well plate for a 7-minute incubation on a rocker in the dark. Then 50 μL of 1% N-1-napthylethylenediamine dihydrochloride (dissolved in water) was added followed by another 7-minute incubation. Optical absorbance was measured at 562 nm and nitrite concentrations in supernatant samples were obtained with reference to a standard curve.
Identification and quantification of rosmarinic acid in P. vulgaris extract were performed with Beckman Coulter System Gold Nouveau HPLC coupled to Beckman System Gold 168 UV/VIS diode array detector (PDA) controlled by 32karat ™ software (Version 5.0) with Supelcosil LC-18 (250×4.6 mm, 5 μm) column (Sigma, St. Louis, MO). The solvent system was 5% acetic acid in water as A and 25% acetonitrile plus 5% acetic acid as B at a flow rate of 1.0 mL/min. The following gradient was used: 40% B/60% A to 70% B/30%A for 10 minutes, then to 100% B for 1 minute, and finally to 40% B/60% A for 12 minutes. Rosmarinic acid concentrations were determined with UV absorbance at 326 nm in comparison with standard compound (Cayman Chemicals, Ann Arbor, MI). Reverse phase analytical YMC-pack ODS C18 (250×4.6 mm, 5 μm) column (YMC, Kyoto, Japan) was used under room temperature for ursolic acid identification and qualification.The mobile phase used for ursolic acid analysis was 1.25 % phosphoric acid and acetonitrile at 0.5 mL/min. The gradient used was:15% acetonitrile at 0 min, then increased to 84% over15 min and held for 40 min, and finally decreased to 15% at 65 min. Ursolic acid concentrations were determined with UV absorbance at 210 nm in comparison with standard compound (Sigma, St. Louis, MO).
For COX-1 and COX-2 measurement, cell lysate was acquired after an 8-hour treatment as previously described (21), followed by SDS-PAGE separation and ECL detection, while iNOS samples were collected after a 24-hour treatment. Mouse monoclonal primary antibodies against COX-1 (sc-19998), iNOS (sc-7271), COX-2 (sc-19999) and α-Tubulin (sc-8035) (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted to 1:1000, 1:600, 1:1200 and 1:2000 individually in 5% powdered milk in Tris buffer saline with 0.1% Tween-20. Arbitrary densities after normalization of four replicate blots were archived by Quantity One program (Bio-Rad, Hercules, CA).
For the cytotoxicity assay, three measurements of each treatment were collected in each of three plates and averaged within plates. PGE2 and NO activities for treatments in each experiment were measured on three plates. For other experiments, a randomized complete block ANOVA was conducted on log-transformed PGE2 concentration, NO concentration, and cell viability values, with plate as fixed block to identify treatment effect. For each assay, a control treatment was included for media and vehicle (water or DMSO) with and without LPS stimulation. All treatments and the water vehicle control were compared against the media+DMSO vehicle control and the averages and standard errors as percentage of vehicle control for each treatment were reported. Multiple comparisons among treatments for Western blotting data were conducted with pair-wise Student t-test after a significant difference was found among treatments by ANOVA (SAS 9.0 SAS Institute Inc, Cary, NC).
All water and ethanol extracts, from all accessions included in this study, were screened for cytotoxicity at 30 μg/mL, the maximal concentration used in this study. In addition, fractions from certain extracts were also screened at the concentrations used in activity assays. Cytotoxicity was not observed with RAW 264.7 macrophages treated with any extract or fraction, while the ursolic acid, as a positive cytotoxic control, caused 50% reduction of cell viability (p<0.01) at 30 μM (150 fold the concentration found in P. vulgaris extracts).
Both water and ethanol extracts from all four 2007 harvested P. vulgaris accessions (Ames 27664, 27665, 27666, 27748) were included in the initial screening for inhibition of PGE2 and NO production at 30 μg/mL concentration, the highest concentration achievable with all extracts. As shown in Figure 1, ethanol extracts from all accessions except Ames 27748 significantly inhibited LPS-induced PGE2 production as compared to the DMSO vehicle control treatment. On the contrary, water extracts did not exert significant inhibition of PGE2 production at the same concentration. Ethanol extracts from Ames 27664, 27665, and 27666 inhibited LPS-stimulated PGE2 by 36%, 23%, and 25%, respectively. LPS-induced NO production was significantly inhibited by all extracts except for the water extract from Ames 27748. Water extracts from Ames 27664, 27665, and 27666 significantly decreased NO level by 10-15%, which were less potent than their ethanol-extracted counterparts, which inhibited NO by 24-26%. Although Ames 27748 water extract had no significant effect on NO production, ethanol extract from this accession decreased NO by 69% as compared to vehicle control. Different treatment strategies, including pretreatment with extracts or LPS, as described in the methods, did not change the observed effect (data not shown). No change in either baseline PGE2 or NO production was seen when RAW 264.7 macrophages were treated with extracts without LPS stimulation (data not shown).
Ethanol extracts from all accessions harvested in 2008 were applied to cell culture at 30 μg/ml concentration. As shown in Table 2, in comparison to the DMSO vehicle control, accessions Ames 27664, 28358, 28357 and 28355 significantly reduced LPS-stimulated PGE2 production, and all extracts significantly decreased LPS-induced NO. Relatively greater overall inhibition of LPS-induced PGE2 and NO release was observed on extracts from accessions Ames 27664, 28358, 28357 and 28355.
Since ethanol extract from accession Ames 27664 harvested in 2007 had the greatest inhibitory activity on LPS-induced PGE2 and NO release among all accessions from both years tested, the dose-response relationship for its activity was evaluated. Four different doses were applied to cells: 30, 20, 15 and 7.5 μg/mL. The resulting effect on LPS-induced inflammatory response is shown in Figure 2, indicating the inhibition on LPS-induced PGE2 and NO production followed a dose-dependent pattern with stronger inhibition accompanied at higher concentration.
Ethanol extracts of accessions Ames 27664, 28358 and 28355 from the 2008 harvest were also applied to cell culture at serial doses to establish the dose-response relationship. For each extract, administration concentrations were 30, 10 and 5 μg/mL. As shown in Figure 3, all three extracts demonstrated a dose-dependent activity in reducing PGE2 and NO production, although the reduction was not statistically significant at lower concentrations. Consistent with the initial screening, these extracts significantly inhibited both LPS-induced PGE2 and NO release at the starting concentration 30 μg/mL. When dose decreased, the inhibition was weakened, as only accessions Ames 27664 and 28355 showed significant effect at 10 μg/mL on PGE2 and NO production, respectively.
Both water and ethanol extracts from accession Ames 27664 (2007 harvest) were fractionated and screened for activity. As Figure 4A and 4B show, at the concentrations proportional to their yield from the ethanol extract, fractions 1, 3, and 5 significantly inhibited LPS-induced PGE2 production, while NO level was reduced by all fractions. When the dose was normalized to 20 μg/mL, only fraction 3 significantly reduced both PGE2 and NO production by 28% and 8%, respectively. Fraction 5 also significantly alleviated NO release by 11%, as indicated by Figure 4C and 4D. In contrast, fractions from the water extract of same plant material did not exert any significant inhibition on LPS-stimulated PGE2 or NO release (data not shown). The baseline PGE2 and NO production without LPS induction were significantly elevated compared to control and other treatments. In order to clarify whether the ethanol extract had active components that were also present in the water extract, a sequential extraction with water was performed on Ames 27664 ethanol extract. The re-extracted product was tested for activity against LPS-induced NO release along with the ethanol extract. While the ethanol extract significantly reduced NO production by 22%, the sequential ethanol then water extraction product had no activity compared to the vehicle control.
Because rosmarinic acid (RA) is a proven anti-inflammatory and anti-oxidative polyphenol carboxylic component of P. vulgaris (8, 22), its abundance was quantified (Table 3). The overall mean ± SE RA content for all accessions was 2.1 ± 0.3 μM in 30 μg/mL extract. Extracts from Ames 27748 and 27664 (2007 and 2008 harvest) contained significantly more RA than did the others, while those from Ames 28356 and 28359 had the lowest RA concentrations. Fractions from Ames 27664 ethanol extracts also contained rosmarinic acid (Table 4). Fractions 5 and 7 had significantly more RA at concentrations in proportion to their yield. When normalized to the same fraction concentration of 20 μg/mL, only fraction 5 contained higher amount of RA. Ursolic acid (UA) concentration is very low in P. vulgaris ethanol extract as we detected 0.2 μM in 30 μg/mL Ames 27664 extract and none in any of its fractions.
The effects of pure RA, ethanol extracts, and extracts enriched with RA were compared on LPS-induced PGE2 and NO production. Pure RA compound was applied to cell culture at 2.67 μM, the concentration found in Ames 27664 ethanol extract from the 2007 harvest. The enrichment dose was also 2.67 μM in order to allow us to assess whether an additive or synergetic effect existed between RA and other components of the extracts. As displayed in Figure 5, RA alone significantly inhibited both LPS induced PGE2 and NO production by 15% and 17%, respectively. Ames 27664 ethanol extract had a significantly stronger inhibition of 31% and 20%, while the ‘RA enriched’ ethanol extract from the same accession inhibited the two inflammation mediators by as much as 39% and 29%. RA enrichment significantly enhanced the potency of the extract in an additive fashion; no interaction between RA and whole extracts was observed (p=0.85) when analyzed with 2×4 2-way ANOVA (RA effect and extracts effect).
After 8-hour treatment with ethanol extracts from Ames 27664 harvested in 2007 and 2008, as well as with pure RA, COX-2 expression in RAW 264.7 macrophages was assayed by Western blot. As shown in Figure 6, P. vulgaris ethanol extracts and pure RA significantly suppressed LPS-stimulated COX-2 expression as compared to media+DMSO vehicle control. There was no significant difference in treatment effect on COX-2 expression except that the 2007 extract was more effective than those from 2008. With regards to iNOS expression, significant inhibition after 24-hour induction was only observed with extracts but not RA treatment, and the quercetin control was more active than the other treatments. At the same time, constitutive expression of COX-1 was not affected by any of the treatments.
Prunella vulgaris has been used as traditional and alternative therapy for minor acute inflammation and chronic inflammatory diseases for over two decades (1), but systematic scientific proof of its efficacy is limited. While the majority of earlier research focused on the immune-regulatory, skin UV-damage protective and anti-microbial effects of P. vulgaris water extracts (4, 23), our study showed that ethanol extracts from selected accessions of P. vulgaris significantly inhibited production of LPS-induced inflammatory mediators PGE2 and NO by RAW 264.7 mouse macrophages. This is in accordance with reports on anti-inflammatory activity of P. vulgaris ethanol extracts (3, 8, 24), while water extract was attributed with putative immunostimulatory activity (4). However, to our knowledge, this is the first study to systematically demonstrate that P. vulgaris ethanol extracts from various accessions dose-dependently inhibited LPS-induced PGE2 and NO production without cytotoxicity. The water extracts from the same accessions had no effect on PGE2 production, but exerted mild inhibition on NO production. A past study of the water extracts used in the present research demonstrated the presence of abundant polysaccharides, associated with anti-viral activity (5). In order to unveil whether polysaccharide was a common active component in both water and ethanol extracts, we tested the effect of aqueous re-extraction of the most active Ames 27664 ethanol extract against LPS-induced PGE2 and NO release, and compared it to the original ethanol extract. This sequential extract did not significantly reduce PGE2 production, suggesting that polysaccharides did not play a role in the anti-inflammatory activity of ethanol extract. We did not further study the water extracts due to their very limited activity in the study. While most assayed ethanol extracts were able to attenuate both the LPS-induced production of PGE2 and NO, their potency on these two major inflammation mediators varied among accessions. This difference suggests that active compound(s) were produced unequally in these plant materials, even in the same accession from different harvest, and highlights the importance of chemical profile in anti-inflammatory activity. So far, accession Ames 27664 exerted the greatest inhibition on LPS-induced PGE2 and NO. In general, P. vulgaris ethanol extracts inhibited LPS-induced PGE2 and NO by 20-40% at concentrations as high as 20-30 μg/mL, which was a lower specific activity than we have seen in parallel studies of Hypericum (13) and Echinacea (14).
Production of PGE2 and NO by macrophages upon LPS induction was reported to be mediated by the toll-like receptor 4 (TLR-4), and subsequent nuclear factor-κB (NF-κB) down-stream activation, which results in expression of COX-2 and iNOS (25). However, we saw somewhat different patterns between the effects of P. vulgaris extracts on PGE2 and NO production. For instance, Ames 27748 ethanol extract had no significant effect on PGE2 but imposed strong inhibition on NO production. This could result from differential effect on transcriptional or/and post-transcriptional regulation of COX-2 and iNOS, which is worthy of future investigation.
Ethanol extract from Ames 27664 was fractionated into seven fractions with the intent of identifying possible active components. Three fractions demonstrated significant inhibition of LPS-induced PGE2 and NO production at concentrations in proportion to their yield from the extract. Among them, fractions 3 and 5 respectively had significant effect on NO when treatment concentration was normalized to 20 μg/mL, while only fraction 3 significantly attenuated PGE2 level at this concentration. These observations revealed that active components of the ethanol extract were distributed in more than one fraction and act through different pathways. Considering that fraction 3 was the only fraction that significantly decreased both the PGE2 and NO production at 20 μg/mL, it was expected to be relatively more abundant in active compounds or contain the major active constituent. On the other hand, fraction 5 could also be an important contributor to the observed extract anti-inflammatory activity, considering both its high yield from extract and its activity at higher concentration.
Rosmarinic acid, a polyphenol compound with known anti-inflammatory and anti-oxidant activity (26, 27), was of interest because of its presence in P. vulgaris ethanol extracts (16). Fraction 5 of the ethanol extract from Ames 27664 showed significant inhibition on both PGE2 and NO production, while it contained the most of the RA in the extract. This suggested RA being a probable anti-inflammatory component in P. vulgaris ethanol extracts. Pure RA treatment at the concentration that occurred in P. vulgaris ethanol extract significantly inhibited LPS-induced PGE2 and NO production, although it did not explain all the extract's activity as shown by the lower inhibitory potency. This confirmed RA as one active compound that partially accounted for the overall anti-inflammatory effect of P. vulgaris ethanol extracts. This is in accordance with the recently published study on human gingival fibroblasts by Zdarilova et al., in which aqueous ethanol extract of P. vulgaris and the corresponding RA component inhibited LPS induced oxidative stress and expression of several pro-inflammatory enzymes including iNOS (8). Compared to their study, we had a lower abundance of RA content (1.73-3.88% of dry matter weight vs. 9.0%) in the extract, which likely resulted from our using 95% ethanol for extraction, instead of 30% ethanol as they used. According to Chizzola et al., aqueous-ethanol extract was more capable of concentrating flavonoids in Thymus vulgaris (28), which is likely the same case for P. vulgaris and may explain the less RA in our extracts. The main reason that we used 95% ethanol was to minimize endotoxin extraction. Although our extract was not designed to enrich RA content, it was intriguing to see RA effect at the extracted concentration. In order to see whether additional RA can further promote the extract activity, we supplemented the extracts with additional RA. As we expected, the RA enriched extracts exhibited a stronger inhibition of the LPS-induced inflammatory response. We used 2-way ANOVA with block to assess whether there was interaction between RA and other components in the extracts. No interaction was found (p=0.85), suggesting the RA effect was additive instead of synergic when it was added to the extracts. We also attempted to relate RA concentration to activity potencies of various accessions, but the resulting Pearson correlation coefficient was not significant for either PGE2 nor NO inhibition (r=0.42, p = 0.22 for PGE2; r=-0.26, p = 0.4652 for NO). This analysis, together with the observation that the Ames 27664 extract from 2007 harvest had stronger activity but lower RA concentration compared to that from 2008 and the most active fraction 3 did not have most RA, further predicted the existence of anti-inflammatory component(s) other than RA in these P. vulgaris ethanol extracts.
Ursolic acid (UA) is a known triterpene component in P. vulgaris plant material (18). UA concentration was 0.2 μM in 30 μg/mL of the Ames 27664 ethanol extracts, far below the threshold for significant anti-inflammatory effect reported by Ryu et al.(10). This UA abundance was comparable to the results reported by Lee et al.that showed UA accounted for 0.05-0.2% dry matter weight of ethanol extracts of 15 different P. vulgaris market samples (29). We did not detect UA in any of the fractions we studied. Despite the low abundance in the extracts, we examined the effect of pure UA compound on LPS-induced PGE2 and NO production by RAW 264.7 macrophages, and did not see any effect at up to 1 μM concentration (data not shown). This ruled out UA as an independent active component of P. vulgaris. We have been using UA as positive control in cytotoxicity assay at 30 μM, but its cytotoxicity effect in the extracts we used is improbable due to the extremely low concentration.
COX-2 protein expression was significantly inhibited by P. vulgaris ethanol extracts and pure RA, while iNOS protein expression was only slightly attenuated by the extracts but not RA. At the same time, COX-1 protein expression was not affected. This evidence suggests inhibition exists at the transcriptional level, possibly through NF-κB signaling pathways. It is likely that there could be compounds other than RA in the extracts inhibiting the expression and/or the activity of the two critical enzymes in inflammation.
Although this study provides evidence for anti-inflammatory activity of P. vulgaris in this well-defined mouse macrophage model, additional research is required to demonstrate that these observations are relevant to human health and to demonstrate that P. vulgaris impacts macrophages in vivo. For example, studies of bioavailability and long-term low-dose effects in vivo are needed, especially since only relatively high concentration of P. vulgaris extract showed significant activity. For rosmarinic acid, bio-availability has been studied in a pig model by Jirovsky et al., who showed that after 91 days of feeding methanol extract of P. vulgaris, RA and RA metabolites (caffeic acid etc.) were found in plasma at over 0.08 μM (30). Notably, this is considerably lower than the 2.67 μM dose we used. Therefore, our study indicated the potential anti-inflammatory activity of P. vulgaris but still awaits further investigation on its long term in vivo impact.
In summary, ethanol extracts of P. vulgaris inhibited LPS-induced PGE2 and NO production in RAW264.7 mouse macrophage cells. This activity was dose-dependent and varied among accessions and harvests. Rosmarinic acid, a polyphenol component of P. vulgaris ethanol extracts, showed independent anti-inflammatory activity when applied to cells at the same concentration as in the ethanol extract. However, RA could only partially explain the overall activity of the extract. Ethanol extracts from P. vulgaris attenuated both COX-2 and iNOS protein expression while pure RA only demonstrated inhibition on COX-2. Further active component identification and bio-availability studies will help reveal more about P. vulgaris as anti-inflammatory dietary supplement.
We give special thanks to members of the Center for Research on Botanical Dietary Supplements of Iowa State University for their continuous support. We also would like to acknowledge the initial studies on P. vulgaris conducted by Dr. Mee-hye Kim of the Korean FDA. We also appreciate the analytical instrumentation support from Dr. Ann Perera, manager of the W. M. Keck Metabolomics Facility, at Iowa State University. This is a journal paper supported by Hatch Act and State of Iowa funds.
This study was supported by the grant P50 AT004155-06 from Office of Dietary Supplements (ODS), the National Center for Complementary and Alternative Medicine (NCCAM) and National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the ODS, NCCAM, or NIH. Mention of commercial brand names does not constitute an endorsement of any product by the U.S. Department of Agriculture or cooperating agencies.