Anti-inflammatory effects of ethanol extracts of Chinese propolis and buds from poplar (Populus×canadensis).

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Anti-inflammatory effects of ethanol extracts of Chinese propolis and buds from poplar (Populus canadensis) Kai Wang a, Jianglin Zhang a, Shun Ping a, Quanxin Ma b, Xuan Chen c, Hongzhuan Xuan d, Jinhu Shi a, Cuiping Zhang a, Fuliang Hu a,n a

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China Laboratory Animal Research Center, Zhejiang Chinese Medical University, Hangzhou 310053, China c Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China d School of Life Science, Liaocheng University, Liaocheng 252059, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 6 February 2014 Received in revised form 7 May 2014 Accepted 21 May 2014

Ethnopharmacological relevance: Propolis is used widely in a number of cultures as a folk medicine and is gaining wider recognition for its potential therapeutic use, due to its wide range of biological properties and pharmacological activities, especially its anti-inflammatory effects. Despite an increasing number of studies focused on the biological activities of propolis together with its botanical sources, studies on Chinese propolis are insufficient. This study was designed to investigate the anti-inflammatory properties of ethanol extracts from Chinese propolis (EECP) and poplar buds (EEPB) from Populus canadensis Moench (Salicaceae family). Materials and methods: Phytochemical analysis of EECP and EEPB was performed via total phenolic and flavonoid content measurements followed by high-performance liquid chromatography (HPLC) analysis. DPPH and ABTS free-radical scavenging methods were used to evaluate their anti-oxidant properties. The anti-inflammatory effects of EECP and EEPB were investigated in vitro by evaluating their modulating effects on the key inflammatory cytokines and mediators in LPS/IFN-γ co-stimulated RAW 264.7 cells and by measuring nuclear factor (NF)-κB activation in TNF-α or IL-1β stimulation HEK 293 cells using reporter gene assays. Their effects on acute inflammatory symptoms (LPS-induced endotoxemia and acute pulmonary damage) were also examined in mice. Results: EECP and EEPB exhibited strong free-radical scavenging activity and significant in vitro antiinflammatory effects by modulating key inflammatory mediators of mRNA transcription, inhibiting the production of specific inflammatory cytokines, and blocking the activation of nuclear factor (NF)-κB. The administration of EECP and EEPB (25 and 100 mg/kg) provided significant protective effects by attenuating lung histopathological changes and suppressing the secretion of LPS-stimulated inflammatory cytokines, such as interleukin-6 (IL-6), IL-10, MCP-1, TNF-α and IL-12p70 production in endotoxemic mice. Conclusions: The results presented here reveal the potent anti-inflammatory properties of Chinese propolis and poplar buds, and provide biological information for developing suitable substitute(s) for propolis in the prevention and treatment of inflammatory diseases. & 2014 Published by Elsevier Ireland Ltd.

Keywords: Chinese propolis Poplar buds Anti-oxidant Anti-inflammatory NF-κB

1. Introduction

Abbreviations: CAPE, caffeic acid phenethyl ester; COX-2, cyclooxygenase (COX)2; DEX, dexamethasone; EECP, ethanol extracts of Chinese propolis; EEPB, ethanol extracts of poplar buds; HPLC, high-performance liquid chromatography; HO-1, heme oxygenase-1; IFN-γ, interferon-γ; IL, interleukin; iNOS, induced nitric oxide synthase; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; NO, nitric oxide; PGE-2, prostaglandins-2; TNF-α, tumor necrosis factor-α; ROS, reactive oxygen species n Corresponding author. Tel./fax: þ 86 571 88982952. E-mail address: fl[email protected] (F. Hu).

Propolis is a resinous substance collected by Apis mellifera from various tree buds for use as a sealant in the hive. Propolis is also widely used in a number of cultures as a folk medicine and is gaining wider recognition for its potential therapeutic use, due to its wide range of biological properties and pharmacological activities (Sforcin and Bankova, 2011). Bees collect the gummy and balsamic materials from buds and resinous exudates of a diverse range of plants over a wide geographic area, leading to substantial differences in the chemical composition and biological activity of propolis (Toreti et al., 2013).

http://dx.doi.org/10.1016/j.jep.2014.05.037 0378-8741/& 2014 Published by Elsevier Ireland Ltd.

Please cite this article as: Wang, K., et al., Anti-inflammatory effects of ethanol extracts of Chinese propolis and buds from poplar (Populus canadensis). Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.037i

K. Wang et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Investigation of the chemical composition of Brazilian green propolis derived from Baccharis dracunculifolia DC (Asteraceae) revealed that it was very similar to that of the parent plant. Both of them contained an abundance of prenylated p-Coumaric acids, mainly artepillin C and baccharin. Comparative studies also found that extracts of Brazilian green propolis and Baccharis dracunculifolia display significant anti-oxidant (Guimaraes et al., 2012), antimicrobial (da Silva Filho et al., 2008), anti-ulcer (Lemos et al., 2007), antileishmanial (da Silva Filho et al., 2009), antimutagenic (Munari et al., 2008), immunomodulatory (Missima et al., 2007) and anti-inflammatory (Cestari et al., 2011; dos Santos et al., 2010) effects both in vitro and in vivo. In Europe and Asia, including China and other temperate zones, the main sources of propolis is the buds of poplar trees (Populus spp., Salicaceae family) (Sforcin and Bankova, 2011; Wu et al., 2008). Propolis from these regions features similar chemical characteristics, with high contents of flavonoids, phenolic acids and their esters (Toreti et al., 2013). The bud exudates of poplar trees and poplar type propolis also have similar qualitative compositions, but they may be very different quantitatively. It has been reported that chrysin, galangin, pinocembrin, quercetin, kaempferol and some phenolic acids (CAPE, p-Coumaric acid, etc.) are the predominant bioactive constituents presented in poplar type propolis and poplar tree buds (Rubiolo et al., 2013). Previous studies have reported that flavonoids and phenolic compounds are the main components responsible for the diverse pharmacological activities of poplar propolis extracts (Volpi and Bergonzini, 2006). Moreover, we have shown that Chinese propolis (poplar type) have strong potential anti-oxidant, anti-inflammatory and anti-diabetic effects both in vitro and in vivo (Wang et al., 2013; Xuan et al., 2011; Zhu et al., 2011), reflecting the abundance and diversity of the flavonoids present in Chinese poplar type propolis (Wu et al., 2008). However, the collection and harvesting process of propolis is slow and costly. According to previous studies, raw propolis production from a single colony varies from 10 to 300 g annually (Sahinler and Gul, 2005), which is much lower than the production rate of the other hive products. The high demand but limited availability of propolis motivates us to find suitable substitute (s) for this valuable natural product. Because various studies found that poplar buds are the botanical origin of Chinese poplar type propolis, we hypothesized that poplar bud extracts may be promising candidates. Additionally, other recent studies have demonstrated that poplar bud extracts have antioxidant, anti-inflammatory, cardiovascular and hepatoprotective effects (Debbache-Benaida et al., 2013; Dudonné et al., 2011). However, until now, studies comparing the biological activities of poplar type propolis as well as poplar bud extracts are still very limited. Thus, in the present study, we compared the biological activities of ethanolic extracts of Chinese propolis (poplar type) and poplar buds (from Populus canadensis Moench, Salicaceae family). These comparisons included in vitro free-radical scavenging activities and anti-inflammatory effects in representative cell lines. We also studied their in vivo anti-inflammatory effects in LPS-challenged mice. Our results revealed for the first time that poplar bud extracts also exerted comparable biological activities to Chinese poplar type propolis and provided biological evidence for developing functional herbal remedies against inflammatory diseases.

2. Materials and methods 2.1. Chemicals DPPH, ABTS, α-tocopherol (Vitamin E), LPS (Escherichia coli 0111:B4), gallic acid, quercetin and the standards used in HPLC analysis were purchased from Sigma (St. Louis, MO, USA).

Recombinant mouse IFN-γ and human TNF-α and IL-1β were purchased from Peprotech (Rocky Hill, NJ, USA). Folin-Ciocalteu reagent, Griess reagent, NaNO2 and dexamethasone were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). Other chemicals were of analytical grade and purchased from Sangon Biotechnology. 2.2. Sample collection and extraction The Chinese propolis samples were obtained from colonies of honeybees, Apis mellifera L., in Shandong, China, in the summer of 2010. The main plant origin was poplar (Populus sp.), belonging to the Salicaceae family. Ethanolic extracts of propolis were prepared using 95% (v/v) ethanol/water and then concentrated in a rotary evaporator under a reduced pressure at 50 1C, as described previously (Wang et al., 2013). Canadian poplar buds (from Populus canadensis Moench, Salicaceae family) were purchased from the Senlei Plant processing company (Changge, Henan, China) and extracted industrially. A voucher specimen (no. 120704) has been deposited at College of Animal Sciences, Zhejiang University. During the cell experiments, the ethanol extracts of propolis (EECP) and poplar buds (EEPB) were redissolved in ethanol and filtered with a 0.22-μm syringe filter to obtain 20 mg/ml stocks and stored at 20 1C in the dark until used. The final concentration of ethanol in the cell medium was less than 0.1% (v/v). For animal experiments, 1 g EECP and EEPB were redissolved in 40 ml 0.5% gum tragacanth to obtain 25 mg/ml stocks, which were stored similarly. The total phenolic and total flavonoid contents of the EECP and EEPB samples were determined by the Folin-Ciocalteu method and aluminum chloride colorimetric method, respectively, as described previously (Ahn et al., 2007). TFC and TFC were expressed as milligram (mg) gallic acid equivalents (GAE)/g and mg quercetin equivalents (QE)/g, respectively. 2.3. High-performance liquid chromatography (HPLC) analysis 2.3.1. Comparing the compositions of propolis and poplar tree buds According to our previous studies, salicin is detectable in poplar extracts but not in propolis. Thus, we used HPLC analysis to distinguish EECP and EEPB as described previously (Zhang et al., 2011). 2.3.2. Separating flavonoids and phenolic acids in EECP and EEPB Chromatographic analyses were carried out on the same Agilent HPLC system. The separation of flavonoids and phenolic acids was performed on Agilent Eclipse XDB-C18 column (4.6 mm 150 mm, 5 μm) at 30 1C. The mobile phases were (A) acetonitrile and (B) 0.4% acetic acid with a flow rate of 1.0 ml/min. The gradient elution was applied as follows: 0–40 min, 5–25% (A); 40–45 min, 25–35% (A); 55–60 min, 35–40% (A); 80–90 min, 40–100% (A). The detection wavelength was 280 nm. All sample solutions were filtered through 0.22-μm membrane filters, and the injection volume was 5 μl. 2.4. In vitro antioxidant activity of EECP and EEPB 2.4.1. DPPH radical-scavenging test Hydrogen-donating activity was measured by direct hydrogen donation to the DPPH radicals as reported previously with some modifications (Ahn et al., 2007). Different concentrations of EECP and EEPB ranging from 10 to 150 μg/ml were prepared for the reaction. The reaction mixtures in the 96-well plates consisted of the sample (100 μl) and DPPH radicals (100 μl, 50 μg/ml) dissolved in ethanol and kept in the dark for 30 min. The absorbance was measured at 517 nm against a blank. All determinations were performed in triplicate. The scavenging activity of the samples was



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expressed as the IC50 value, the concentration required to scavenge 50% of DPPH radicals. 2.4.2. ABTS radical-scavenging test The ABTS radical-scavenging activity assay was carried out by ABTS cation radical decolorization with minor modifications (Shi et al., 2012). The EECP and EEPB solutions were prepared as described for the DPPH assay. The ABTS cation radical was prepared by reacting a 7 mM aqueous solution of ABTS (15 ml) with 140 mM potassium persulphate (264 μl). The mixture was allowed to stand in the dark at room temperature for 16 h before use. Prior to the assays, the ABTS working reagent was diluted with methanol to give an absorbance of 0.70 70.02 at 734 nm after equilibration at room temperature. The reaction mixtures in the 96-well plates consisted of the sample (50 μl) and the ABTS methanol working solution (100 μl). The mixture was stirred allowed and left to stand for 10 min in the dark, after which the absorbance was measured at 734 nm against a blank. All determinations were performed in triplicate. The scavenging ability of the samples was expressed as the IC50 value, the effective concentration at which 50% of the ABTS radicals were scavenged. 2.5. Cell culture and cell viability assay Murine macrophage RAW 264.7 cells, human embryonic kidney (HEK) 293T and HEK-293 cells stably expressing the IL-1R (C6 cells) cells were used. These were a generous gift from Professor Zongping Xia (Life Sciences Institute, Zhejiang University, China). The cells were incubated in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 1C in a 5%-CO2 atmosphere. The cell viability assay was performed using a CCK-8 kit (cell counting kit-8) (Dojido, Japan). According to the manufacturer’s instruction, 10 104/ml cells were seeded into 96-well cell culture plates. After 24 h incubation, the RAW 264.7 cells and HEK 293T cells were incubated in the presence of various concentrations of EECP or EEPB. Next, 24 h later, the cells in each well were incubated with 10 μl of CCK-8 at 37 1C for 2 h. The optical density (OD) for each well was then measured at 450 nm using a microplate reader (Bio-Rad Model 550, CA). Cell viability was also confirmed by trypan blue exclusion and microscopy during the following experiments. 2.6. RNA isolation and quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) The total cellular RNA from RAW 264.7 cells was isolated using an RNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's instructions and stored at 80 1C until used. One microgram of total RNA was used as a template for first-strand cDNA synthesis using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The primers are presented in Table 1 and were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Quantitative real-time PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) with a SYBR Premix Ex Taq (TaKaRa, Dalian, China) following the manufacturer’s instructions via a standard two-step PCR reaction. Specificity was confirmed by dissociation curve analysis and gel electrophoresis. GAPDH was used as a housekeeping gene to normalize the expression of the target genes, and the results were expressed as 2 ΔΔCt (Livak and Schmittgen, 2001). 2.7. Cell transient transfection and reporter gene assay The HEK 293T or 293 C6 cells were plated at 1 105 cells/well (1 ml culture volume) of a 12-well plate (Corning, USA) in DMEM plus 10% FBS. After overnight incubation, 30 ng firefly luciferase

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Table 1 Primer sequences used for qRT-PCR experiments. Gene

Primer sequence

Product size (bp)

GenBank accession no.

GAPDH

50 -GAGAAACCTGCCAAGTATGATGAC-30 50 -TAGCCGTATTCATTGTCATACCAG-30 50 -CTCTGCAAGAGACTTCCATCC-30 50 -GAATTGCCATTGCACAACTC-30 50 -CTATGCTGCCTGCTCTTACTG-30 50 -CAACCCAAGTAACCCTTAAAGTC-30 50 -TTTCCAGAAGCAGAATGTGACC-30 50 -AACACCACTTTCACCAAGACTC-30 50 -GAAATATCAGGTCATTGGTGGAG-30 50 -GTTTGGAATAGTTGCTCATCAC-30 50 -CCACGCTCTTCTGTCTACTG-30 50 -ACTTGGTGGTTTGCTACGAC-30 50 -ACATTGAGCTGTTTGAGGAG-30 50 -TACATGGCATAAATTCCCACTG-30 50 -AAGAAGCTGTAGTTTTTGTCACCA-30 50 -TGAAGACCTTAGGGCAGATGC-30 50 -GGCAGCAGATGGAAAACCT-30 50 -AGGGCTTTCTGCTCAGGTCT-30

212

NM_008084.2

210

NM_031168.1

221

NM_010548.2

294

NM_010927.3

237

NM_011198.3

169

NM_013693.2

241

NM_010442.2

155

NM_011333.3

183

NM_009971.1

IL-6 IL-10 iNOS COX-2 TNF-α HO-1 MCP-1 G-CSF

reporter plasmid (pGL4.2-3 NF-κB-Luc) and 5 ng sea pansy luciferase reporter plasmid (pRL-TK) were transfected using polyethylenimine (PEI, Polysciences Inc.) into the HEK 293T or 293 C6 cells together with the pcDNA3.1 vector to yield 500 ng of expression plasmids in total. Next, 12 h after transfection, HEK 293T or 293-C6 cells were pretreated with various concentrations of EECP/EEPB for 1 h. HEK 293T cells were stimulated with recombinant TNF-α and 293-C6 cells with recombinant IL-1β for another 12 h at a final concentration of 10 ng/ml. Finally, the cells were collected, and the firefly and sea pansy luciferase activities were measured using a DLReady luminometer (Berthold Technologies, Germany) with the firefly luciferase activity normalized for transfection efficiency based on the sea pansy luciferase activity, as previously described (Wang et al., 2013).

2.8. Determination of nitric oxide (NO) production NO levels were determined in the cell medium using the Griess reaction. Specifically, 100 μl of supernatant from each well was mixed with 100 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine in 2.5% phosphoric acid) in a separate 96-well plate. After a 10-min incubation at room temperature, the optical density was determined at 540 nm with a microplate reader. A standard curve using NaNO2 was generated for quantification in each experiment.

2.9. Animal experiments Male ICR mice (6–8 weeks old, 18–22 g) were purchased from the Animal Experimental Center, Zhejiang Chinese Medical University, China. They were housed in cages (8 mice per cage) in a room with controlled lighting (12 h light/dark cycle) and temperature (20–23 1C). The air supply was filtered and pathogen-free air, with a relative humidity of 50%. The mice were allowed free access to a commercial, non-purified diet and water. The experiments reported in this study were carried out in accordance with local guidelines for the care of laboratory animals of the Animal Experimental Center, Zhejiang Chinese Medical University, and were approved by the university's ethics committee for research on laboratory animals. The numbers of laboratory-animal-quality certification and experimental facility license were SYXK(ZHE) 2008-0115 and SCXK(HU)2008-0016, respectively.



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2.9.1. Animal groups, drug administration and induction of endotoxemia and acute lung injury (ALI) After 1 week of adaptation, the mice were randomly divided into seven groups (n ¼8 each): (1) the standard group, in which the mice received the vehicle as a normal control; (2) the model group, in which the mice were injected LPS (1 mg/kg) through the tail vein as a negative control; (3) the positive control group, in which dexamethasone (DEX, 2 mg/kg) was injected through the tail vein 1 h before LPS challenge; (4) the EECP low-dose group, in which the mice were orally administered 25 mg/kg EECP with tailvein-injected LPS; (5) the EECP high-dose group, in which the mice were orally administered 100 mg/kg EECP with tail-vein-injected LPS; (6) the EEPB low-dose group, in which the mice were orally administered 25 mg/kg EEPB with tail vein injected LPS; and (7) the EEPB high-dose group, in which the mice were orally administered 100 mg/kg EECP with tail-vein-injected LPS. For three days in a row, the mice were orally administered with EECP and EEPB (25, 100 mg/kg) for pretreatment, while the other groups (standard, model and positive control groups) received the vehicle control. On the third day, 1 h after the final EECP/EEPB administrations, all of the animals (except for the standard groups) were injected with LPS through the tail vein to induce endotoxemia and acute lung injury (Choi et al., 2013).

3. Results and discussion 3.1. Phytochemical analysis of EECP and EEPB Phenolic and flavonoid compounds are considered to contribute most to the therapeutic effects of propolis; thus, the phytochemical analysis of EECP and EEPB are important for the biological activity studies. We found that the total flavonoid content (TFC) of the EEPB was comparable to that of EECP (Table 2). Interestingly, the total phenolic content (TPC) of EECP was much higher than that of EEPB. According to previous studies, the amounts of TPC and TFC in Chinese poplar propolis varied from 42.9 to 302 mg GAE/g of TPC and from 8.3 to 188 mg QE/g of TFC. Our Chinese propolis extracts and the poplar buds extracts used in the present study were comparable to the literature values, being at the relatively high ends of these ranges (Ahn et al., 2007). According to our previous studies, salicin can be used as a marker to distinguish EEPB from EECP (Zhang et al., 2011). Salicin was found in poplar tree bud extracts but not in propolis extracts because it could be hydrolyzed during propolis collection by bees

2.9.2. Histopathology examination Lung specimens were fixed in 10% formalin and then embedded in paraffin. From six to ten 4-μm-thick sections were prepared in a noncontiguous way and stained with hematoxylineosin (HE). The HE-stained slides were visualized under a light microscope (Nikon eclipse 80i) and images were acquired using the attached camera (Zhu et al., 2011). 2.10. Inflammatory cytokine measurement of cultured cells and mice serum Culture supernatants from the RAW 264.7 cells and serum from the mice were harvested for cytokine measurement using a cytometric bead array (CBA) mouse inflammation kit (BD Biosciences, Cat no. 552364) according to the manufacturer's guidelines for detecting IL-6, IL-10, MCP-1, IFN-γ, TNF and IL-12p70 production, simultaneously. Culture supernatants were diluted 50 times and serum samples were diluted for 25 times as necessary. The samples were analyzed by a FACSCalibur apparatus using FCAP Array software (BD Biosciences). 2.11. Statistical analysis Data are expressed as the means 7SD for the indicated number of independently performed experiments. Statistical comparison of the data was performed by Student’s t test or one-way ANOVA using the Student–Newman–Keules method. p-Values less than 0.05 were considered statistically significant. All statistical tests were carried out using SPSS 17.0.

Fig. 1. HPLC chromatograms for (A) salicin and (B) other selected phenolic acids and flavonoids in EECP (red line) and EEPB (green line). Peaks represented: S, salicin; A, unknown compound(s); (1) caffeic acid; (2) p-Coumaric acid; (3) ferulic acid; (4) resveratrol; (5) quercetin; (6) apigenin; (7) kaempferol; (8) chrysin; (9) pinocembrin; (10) galangin; (11) CAPE. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Total phenolic content (TPC), Total flavonoid content (TFC) and DPPH/ABTS free-radical scavenging activities of EECP and EEPBa. Sample

EECP EEPB α-Tocopherol a

TPC (mg GAE/g)

233.987 0.84 145.54 75.89 –

TFC (mg QE/g)

124.92 7 9.74 126.23 78.46 –

IC50 (μg/ml) DPPH-scavenging activity

ABTS-scavenging activity

15.497 0.59 28.69 7 1.52 17.80 7 1.11

36.66 71.82 55.63 70.78 56.43 75.75

Values are the means 7 SD (n¼3). EECP, ethanol extracts of Chinese propolis; EEPB, ethanol extracts of poplar buds.



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(Zhang et al., 2011). As shown in Fig. 1(A), salicin can be clearly detected in EEPB but not in EECP at a wavelength of 213 nm. Meanwhile, peak A, an unknown compound(s) usually detected in poplar extracts, can also be found near after the salicin peak. Therefore, we can infer that our samples can be regarded as good representatives of Chinese propolis extracts and poplar bud extracts. Moreover, we know that the biological activities of propolis and poplar bud extracts were largely the result of various natural polyphenol compounds. Thus, we identified some flavonoids and phenolic compounds by comparison with 11 authentic standards previously found in both EECP and EEPB (Rubiolo et al., 2013; Vardar-Ünlü et al., 2008; Volpi and Bergonzini, 2006). The HPLC separation of EECP and EEPB at 280 nm is shown in Fig. 1(B), and their relative concentrations are shown in Table 3. As found in previous studies, poplar bud extracts and propolis extracts are frequently similar in qualitative composition but may differ in the relative contributions of their various components (Vardar-Ünlü et al., 2008). Chrysin, pinocembrin and galangin are three leading flavonoids present in both EECP and EEPB. Nevertheless, some phenolic acids (caffeic acid, p-Coumaric acid and ferulic acid) could not be detected in EEPB, in contrast to previous reports (Dudonné et al., 2011; Rubiolo et al., 2013). This may explain the discrepancies in the total phenolic contents of EECP and EEPB.

3.2. in vitro antioxidant activity of EECP and EEPB by DPPH and ABTS free-radical scavenging activity assays DPPH and ABTS free-radical scavenging activity assays are two of the most commonly used methods for screening the antioxidant Table 3 Major phenolic acids and flavonoids presented in EECP and EEPBa. Peak no.

1 2 3 4 5 6 7 8 9 10 11

Compounds

Caffeic acid p-Coumaric acid Ferulic acid Resveratrol Quercetin Apigenin Kaempferol Chrysin Pinocembrin Galangin CAPEc

Retention time (min)

17.54 24.36 27.75 40.53 46.24 49.89 50.78 66.56 69.16 70.04 71.76

Contents (g/100 g of extract) EECP

EEPB

0.53 7 0.01 0.447 0.00 0.187 0.00 1.45 7 0.01 0.147 0.01 0.42 7 0.09 3.107 0.10 10.117 0.12 3.65 7 0.16 3.217 0.24 2.337 0.19

NDb ND ND 0.38 7 0.00 1.107 0.02 0.54 7 0.01 1.85 7 0.23 4.177 0.15 2.88 7 0.12 2.83 7 0.21 1.23 7 0.12

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activity of natural products and plant extracts due to the speed, reliability and reproducibility of the assay (Dudonne et al., 2009). Propolis has been identified as a good free-radical or active oxygen scavenger (Ahn et al., 2007), while a recent study reported that poplar buds aqueous extracts exhibit good anti-oxidant activities (Rubiolo et al., 2013). As shown in Table 2, our results indicate that the IC50 value of EECP was comparable to that of α-tocopherol (Vitamin E), a well-known anti-oxidant in the DPPH radical scavenging assay. Additionally, the IC50 value of EEPB suggested that EECP has stronger DPPH-scavenging ability than EEPB. On the other hand, the ABTS assay results indicated that both EECP and EEPB possessed significant radical scavenging activity, equivalent to that of αtocopherol. The presence of phenolic acids and flavonoid compounds in plant materials contribute significantly to their antioxidant potential. Our results are in agreement with previous reports that both EECP (Ahn et al., 2007) and EEPB (Debbache-Benaida et al., 2013) have potent antioxidant properties against free radicals from DPPH or ABTS. However, EECP exerted a more potent DPPH-scavenging ability, which we hypothesized may be related to its more abundant phenolic compounds contents. Indeed, phenolic acids are composed of aromatic rings bearing one or more hydroxyl groups and therefore can therefore potently quench free radicals by forming resonancestabilized phenoxyl radicals (Dudonne et al., 2009). 3.3. Effects of EECP and EEPB on RAW 264.7 and HEK 293T cell viability To ensure that EECP and EEBP do not have toxic effects on cell metabolism and determine the optimal concentrations for the following cellular experiments, the effects of EECP and EEPB on cell viability in RAW 264.7 cells and HEK 293T cells were assessed using CCK-8 assay. Fig. 2 shows the cell viability results after 24 h of incubation using different concentrations of Chinese propolis and poplar bud extracts. Statistically significant decreases in RAW 264.7 cell survival was detected at concentrations up to 10 μg/ml for EECP and up to 20 μg/ml for EEPB (Fig. 2A). For HEK 293 cells, treatment with Chinese propolis extract and poplar bud extract (5, 15, 30 and 50 μg/ml) for 24 h did not cause any significant viability changes relative to the control group (Fig. 2B). The cytotoxic concentrations of Chinese propolis extract and poplar bud extract were much higher for HEK 293T cells than RAW 264.7 cells, suggesting that the mouse leukemia cells (RAW 264.7 cells) are more sensitive to EECP or EEPB than the normal HEK 293T cells. Based on these results, proper concentration ranges were chosen for treatment in the subsequent experiments. 3.4. in vitro anti-inflammatory effects of EECP and EEPB

a

Values are the means 7SD (n¼ 3). b ND, not detected. c CAPE, caffeic acid phenyl ester.

Macrophages play key roles in innate immunity and activating macrophages, initiating and amplifying a variety of inflammatory

Fig. 2. Effects of EECP and EEPB on the cell viability of RAW 264.7 cells and HEK 293T cells. RAW 264.7 cells (A) and HEK 293T (B) cells were treated with various concentrations of EECP or EEPB for 24 h, and cell viability was measured by CCK-8 assay, as described in Section 2. Reported values are the mean 7 SD of at least three separate experiments. np o0.05 versus EECP/EEPB-untreated control cells by Student’s t-test.


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diseases (Chawla et al., 2011). Inhibiting improper macrophage activation or counteracting the overproduction of macrophage products selectively has been suggested as a promising therapeutic route against various inflammatory conditions (Duffield, 2003). RAW 264.7 cells are Abelson murine leukemia virus transformed monocytes/macrophages, which are immunologically active and respond to many inflammatory stimuli (Ndiaye et al., 2012). We first chose LPS (200 ng/ml) combined with IFN-γ (10 ng/ml) to establish a well-accepted model in the following in vitro antiinflammatory studies.

3.4.1. Effects of EECP and EEPB on the mRNA expression of key inflammatory-mediators and cytokine genes in LPS/IFN-γ costimulated RAW 264.7 cells To evaluate the effects of EECP and EEPB on mRNA expression during LPS/IFN-γ co-stimulation in RAW 264.7 cells, eight key genes involved in the inflammatory response were chosen and measured by quantitative real-time PCR. The RAW 264.7 cells were co-stimulated with LPS/IFN-γ or LPS/IFN-γ and EECP or EEPB for 6 h. This time period was chosen based on our preliminary studies as that for the maximal mRNA expression of these inflammatory related genes after the co-stimulation. The pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α as well as the anti-inflammatory cytokine IL-10 are highly expressed during the inflammation process (Szliszka et al., 2013a,, 2013b). Additionally, the activation of induced nitric oxide synthase (iNOS) may cause NO accumulation in the cell supernatant. Cyclooxygenase (COX)-2 is the ratelimiting enzyme involved in the synthesis of prostaglandin E2 (PGE-2) collectively enhancing the inflammatory responses (Rossi et al., 2002; Song et al., 2002). Moreover, heme oxygenase (HO)-1 expression during the inflammation may suppress the inflammatory response by decreasing the production and reducing the effectiveness of NO (Rubiolo et al., 2013). Activated macrophages also express high levels of monocyte chemoattractant protein 1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF), which attract blood monocytes and tissue macrophages and are therefore involved in chronic inflammatory disorders (Remppis et al., 2010). Hence, the mRNA expression levels for these key enzymes and cytokines are important indicators of anti-inflammatory activity. LPS/IFN-γ caused significant changes in the transcription of several inflammatory-related genes, including IL-6, IL-10, iNOS, COX-2, TNF-α, HO-1, MCP-1 and G-CSF(Fig. 3A–H). Among these, pretreatment with ethanol extracts from propolis (5 and 10 μg/ml) and poplar tree buds (10 and 20 μg/ml) decreased IL-6, IL-10, iNOS, COX-2 and MCP-1 mRNA expression (Fig. 3A–C and G) compared to the LPS/IFN-γ co-stimulation group (p o0.05). Additionally, low doses of EECP (5 μg/ml) and EEPB (10 μg/ml) slightly inhibited mRNA expression of TNF-α, an important pro-inflammatory cytokines. Nevertheless, compared with the LPS/IFN-γ co-stimulation group, EECP and EEPB caused dramatic increases in TNF-α mRNA transcription by factors of 3.25 70.8 and 2.73 70.5 when cotreated with high doses of EECP (10 μg/ml) and EEPB (20 μg/ml), respectively. Similar effects appear to occur with G-CSF mRNA expression. However, EECP or EEPB pretreatment alone did not cause any significant mRNA expression of these genes (data not shown). More interestingly, we observed that the co-exposure to EECP, but not EEPB, caused significant increases in HO-1, one of the important antioxidant enzymes that modulate the intracellular ROS level (Min et al., 2012), we also found that HO-1 mRNA transcription was dramatically increased by EECP treatment alone (data not shown). These results suggest that EECP and EEPB might exert potent anti-inflammatory effects and that their antiinflammatory properties might be selective for mRNA transcription during the inflammatory process.

3.4.2. Effects of EECP and EEPB on the production of NO and inflammatory-related cytokines To explore whether EECP and EEPB affected inflammatory mediation, NO production was measured by Griess reaction, and some inflammatory related cytokines were determined using a cytometric bead array (CBA) kit. Nitric oxide (NO) is an important transmembrane molecular signal during the inflammation process (Nathan and Xie, 1994). In accordance with previous reports, NO production is sharply increased for LPS combined with IFN-γ stimulation (Szliszka et al., 2013b). By 24 h after stimulation, NO production in the cell medium had increased to 46.7070.25 μM. EECP and EEPB pretreatment for 1 h significantly inhibited NO production. Moreover, the inhibitory effects of EECP were stronger than those of EEPB. EECP at 10 μg/ml exerted the greatest inhibition, decreasing NO production to 12.75 70.10 μM. EECP and EEPB alone did not significantly increase NO production (p 40.05 compared with the no-stimulation group). The inflammatory cytokines in the cell medium were analyzed using the BD cytometric bead array (CBA), which can analyze multiple cytokines contemporaneously and has comparable analytical sensitivity to conventional ELISAs (Fig. 3B–E). in vitro costimulation with LPS/IFN-γ dramatically enhanced the production of IL-6, IL-10, MCP-1 and TNF-α. Both EECP and EEPB co-treatment were significantly active in neutralizing IL-6 and MCP-1(Fig. 3B and D). Moreover, we found that low dose of EECP (5 μg/ml) promoted the production of IL-10, an important anti-inflammatory cytokine whereas EEPB (10 and 20 μg/ml) and high dose of EECP (10 μg/ml) significantly blocked the production of IL-10 compared to the LPS/IFN-γ co-stimulation group (po 0.05) (Fig. 3C). However, TNF-α production was not affected by EECP or EEPB pretreatment (Fig. 3E). Previous studies indicated that propolis was anti-inflammatory via inhibiting NO release and modulating cytokines production (Bufalo et al., 2013; Szliszka et al., 2013a). It was also demonstrated that EECP could block NO, IL-1β and IL-6 production in LPSactivated RAW 264.7 cells (Wang et al., 2013). Recently, Szliszka et al. (2013a) used Brazilian green propolis to study its in vitro antiinflammatory effects in LPS/IFN-γ stimulated murine macrophage cells and found that EEBP significantly downregulated the production of cytokines including IL-1β, IL-6, IL-12p40 and TNF-α, chemokines, including MCP-1 and G-CSF, in LPS þIFN-γ treated murine J774A.1 macrophages. It must be emphasized that any dose-dependence could well be selective for specific inflammatory mediators. Thus, while poplar bud extracts exerted potential in vitro anti-inflammatory effects similar to their propolis products, the effective concentration of EEPB (10 and 20 μg/ml) used was twice as high as that of EECP(5 and 10 μg/ml). The difference is attributable to discrepancies of the active compounds (flavonoids and phenolic acids, etc.) (Fig. 4). Q2

3.4.3. Inhibition nuclear factor (NF)-κB of EECP and EEPB in TNF-α stimulated HEK 293T cells and IL-1β stimulated 293 C6 cells NF-κB regulates over 100 genes, including inflammatory cytokines, chemokines, immunoreceptors and other cell adhesion molecules (Lawrence, 2009). We evaluated the effects of EECP and EEPB on the NF-κB activation using reporter gene assays using a pGL4.2-3 NF-κB-Luc plasmid. This was generated by inserting three spaced NF-κB binding sites into the pLuc-promoter vector. The exposure of cells to TNF-α leads to the activation of the transcription factors NF-κB engaged signal-transducing molecules through TNFR (Lawrence, 2009). Alternatively, IL-1β or LPS simulation also leads to the activation of NF-κB mediated by toll-like receptor/interleukin-1 receptor (TLR/IL-1R) (Kim et al., 2012). TNFα or IL-1β treatment of the transfected cells increased the NF-κB expression by factors of about approximately 160.1 711.7 and



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Fig. 3. Effects of EECP and EEPB on the mRNA expression of key inflammatory-mediators and cytokine genes in LPS/IFN-γ co-stimulated RAW 264.7 cells. RAW 264.7 cells were pre-treated with various concentrations of EECP (5 and 10 μg/ml) or EEPB (10 and 20 μg/ml) for 1 h before incubation with LPS (200 ng/ml) and IFN-γ (10 ng/ml) for 6 h. The level of mRNA expression of genes is expressed as fold-change relative to the LPS/IFN-γ group. The mRNA expression of IL-6 (A), IL-10 (B), iNOS (C), COX-2 (D), TNF-α (E), HO-1 (F),MCP-1 (G) and G-CSF (H) was analyzed by qRT-PCR. Data are presented as the means 7SD from three independent experiments. One-way ANOVA with the Student–Newman–Keuls method was performed to compare each group, and the means with different letters are significantly different (p o 0.05).


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Fig. 4. Effects of EECP and EEPB on the production of NO and inflammatory-related cytokines in LPS/IFN-γ co-stimulated RAW 264.7 cells. RAW 264.7 cells were pre-treated with various concentrations of EECP (5 and 10 μg/ml) or EEPB (10 and 20 μg/ml) for 1 h before incubation with LPS (200 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitrite oxide (A) content was measured using the Griess reaction and IL-6 (B), IL-10 (C), MCP-1 (D), TNF (E) concentration was measured in culture media using a BD-cytometric bead array (CBA). Each value indicates the mean 7standard deviation (SD) of three independent experiments. One-way ANOVA with the Student–Newman–Keuls method was performed to compare each group, and the means with different letters are significantly different (p o0.05).

Fig. 5. Effects of EECP and EEPB on the activation of NF-κB in TNF-α stimulated HEK 293T cells and IL-1β stimulated 293 C6 cells. HEK 293T (A) or 293-C6 (B) cells were transiently transfected with firefly luciferase reporter plasmid (pGL4.2-3 NF-KB-Luc) and luciferase reporter plasmid (pRL-TK) for 24 h. Cells were pretreated with an different concentrations of EECP (0–50 μg/ml) or EEPB (0–50 μg/ml) for 1 h, and then HEK 293T cells were stimulated with recombinant TNF-α (10 ng/ml) and 293-C6 cells were stimulated with recombinant IL-1β (10 ng/ml) for another 12 h. Luciferase activity was normalized to tk-Renilla luciferase activity, and the results are expressed as relative NF-κB fold changes compared to untreated cells. Data represent the mean 7SD of three independent experiments. Individual groups were compared using Student’s t test (np o0.05, nnpo 0.01, nnnp o 0.001 compared with the TNF-α or IL-1β stimulation group; ♯p o 0. 05 compared with the untreated group).

5.170.3 over the basal level, respectively. However, the treatment of cells with EECP and EEPB significantly inhibited pro-inflammatory cytokine induced NF-κB activation, corresponding to reductions to 37.173.7% and 46.870.3% at 50 μg/ml for EECP and EEPB in TNF-α stimulated HEK 293T cells and 44.371.0% and 45.574.7% in IL-1β stimulated 293-C6 cells, respectively (Fig. 5). To the best of our knowledge, this is the first report about the effects of poplar buds on the NF-κB activation and subsequent gene expression.

3.5. in vivo anti-inflammatory effects of EECP and EEPB The pathogenesis of LPS is attributable, at least in part, to dysregulated systemic inflammatory responses, significant

inflammatory damage in tissues and organs as well as the excessive accumulation of various inflammatory cytokines (Choi et al., 2013). To examine effects of EECP and EEPB on LPS-induced endotoxemia and acute lung injury (ALI) in mice, we measured serum inflammatory cytokines and pulmonary histopathology. When mice were injected with LPS (1 mg/kg for 3 h), morphological changes in pulmonary tissue included increased alveolar wall thickness, edema, bleeding and inflammatory cell infiltrates (Fig. 6B). As shown in Fig. 6D and E, pretreatment with 100 mg of EECP or EEPB/kg of body weight significantly inhibited LPSinduced histological changes. This is even comparable to the effects of 2 mg/kg DEX (Fig. 6C). Moreover, after LPS (1 mg/kg) challenge, serum IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 were higher (Table 4). A representative serum cytokine assay in



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Fig. 6. Effects of EECP and EEPB on LPS-induced lung histopathologic changes. Lungs from each experimental group collected at 3 h after LPS challenge were processed for histological evaluation: lung sections from the (A) standard group; (B) LPS-induced acute lung injury (ALI) model group; (C) mice exposed to LPS and treated with dexamethasone (2 mg/kg). (D and E) mice exposed to LPS and treated with 100 mg/kg of EECP and EEPB, respectively. Representative histological sections of the lungs were stained by hematoxylin and eosin (magnification 200 ).

Table 4 Effect of EECP and EEPB on serum levels of inflammatory cytokines in LPS-challenged micea. Group

Dose (mg/kg)

IL-6 (ng/ml)

IL-10 (pg/ml)

MCP-1 (ng/ml)

IFN-γ (ng/ml)

TNF-α (pg/ml)

IL-12p70 (pg/ml)

Standard Model EECP EECP EEPB EEPB Dexamethasone

– – 25 100 25 100 2

ND 96.007 10.66a 103.277 2.54a 70.15.007 8.58b 88.28 7 2.18a 68.777 3.00b 52.05 7 7.37b

ND 237.15 70.59a 245.18 75.22a 107.99 73.40b 186.16 713.51c 302.12 74.91d 180.22 711.53c

ND 98.277 10.69a 87.137 3.12a,b 82.417 5.04b,c 83.23 7 0.01a,b,c 73.03 7 2.45b,c 67.93 7 1.18c

ND 5.22 7 0.32a 7.58 7 0.16b 10.167 1.05c 3.98 7 0.09a,d 3.36 7 0.05d 2.69 7 0.64d

ND 1897.577 93.31a 2706.41 736.23b 1303.187 113.43c 1981.88 7 93.87a 1263.79 7 74.56c,d 850.82 7 58.44d

ND 100.60 7 8.08a 97.29 7 3.39a,b 67.56 7 8.00b 114.197 25.06a 61.91 71.69b 6.61 70.95c

a Values are the means 7 SD (n ¼8 for each group). One-way ANOVA with the Student–Newman–Keuls method was performed to compare each group, and the means with different letters are significantly different (p o 0.05). ND, not detected.

LPS-challenged mice serum is shown in Fig. 7. We found that EECP (25 and 100 mg/kg) decreased IL-6, IL-10, MCP-1, TNF-α and IL-12p70 production but enhanced the release of IFN-γ. Meanwhile, EEPB (25 and 100 mg/kg) significantly decreased IL-6, IL-10, MCP-1, TNF-α and IFN-γ production, whereas a transient increase in the anti-inflammatory cytokine, IL-10, was observed for 100 mg/kg EEPB treatment. Additionally, the positive control, dexamethasone (2 mg/kg) significantly decreased all of the inflammatory cytokines measured (p o0.05). Our previous studies demonstrated that ethanol propolis extracts exerted great anti-inflammatory effects in vivo using different animal models (Hu et al., 2005). We also found that Chinese propolis could significantly inhibit the increase of IL-6 in inflamed tissues, but had no significant effect on levels of IL-2 and IFN-γ (Hu et al., 2005). Recently, Debbache-Benaida et al. (2013) demonstrated that Populus nigra ethanol extract exerted potent anti-inflammatory activity on carrageenan-induced mice paw edema. Moreover, Machado et al. used LPS-challenged mice model and demonstrated that Brazilian green propolis extract has antiinflammatory properties by lowering proinflammatory cytokines (IL-6 and TNF) and increasing of anti-inflammatory cytokines (IL-10 and TGF-β) production (Machado et al., 2012). All of these studies together with the present study clearly show that EECP and EEPB exert anti-inflammatory effects in vivo by inhibiting the

production of specific inflammatory cytokines, and the efficiency may result from the synergistic effect of the presence of flavonoids and phenolic acids. More interestingly, oral treatment with high dosages of poplar buds (100 mg/kg) also exerted very similar inhibitory effects when compared to Chinese propolis extracts (100 mg/kg) against some pro-inflammatory cytokines production, such as IL-6, MCP-1, TNF-α and IL-12p70.

4. Conclusions In summary, our in vitro and in vivo data support the potent antiinflammatory properties and free-radical scavenging activities of propolis and bud extracts from Chinese poplars. The HPLC fingerprints indicated that EECP and EEPB have similar chemical constituents in different relative compositions. We also provided biological evidence that EEPB, similarly to EECP, presents potent anti-inflammatory and free-radical scavenging activity, suggesting that poplar bud extracts may well be a potential substitute for propolis in the prevention and treatment of inflammatory diseases. In addition, we have shown that the anti-inflammatory effects of EECP and EEPB are mainly due to the modulation of specific inflammatory mediators in LPS/IFN-γ costimulated RAW 264.7 cells and LPS challenged mice. Although


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Fig. 7. Representative cytometry bead array (CBA) assays of inflammatory cytokines in the mice serum. Serum samples from each experimental group collected at 3 h after LPS challenge were processed for CBA assays: (A) the standard group; (B) the model group (1 mg/kg LPS); (C) the mice exposed to LPS and treated with dexamethasone (2 mg/kg); (D and E) the mice exposed to LPS and treated with 100 mg/kg of EECP and EEPB, respectively.



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inhibition of transcription factor NF-κB activity was observed, we cannot exclude the possibility that EECP and/or EEPB may interact with different targets, which will require further studies in both animals and humans.

Funding sources This work was supported by the grant from the National Natural Science Foundation of China (no. 31272512), the Modern Agro-industry Technology Research System from the Ministry of Agriculture of China (CARS-45). Generous support was provided also by the Science and Technology Plan Projects of Zhejiang Province (no. 2013F10001) and National Natural Science Foundation of China (no. 31201860).

Acknowledgment We are grateful to Dr. David Topping from Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia for reviewing our manuscript and giving us valuable suggestions. We also thank to Prof. Zongping Xia from Life Science Institute, Zhejiang University for his generous provision of cell lines. We appreciate the excellent technical support of Dr. Xiaolu Jin from College of Animal Sciences, Zhejiang University.

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Comparisons of ethanol extracts of chinese propolis (poplar type) and poplar gums based on the antioxidant activities and molecular mechanism.

Water Solvents on Phenolic Profiles and Antioxidant Properties of Beijing Propolis Extracts.

Chemicals found in poplar-type propolis.

Inhibitory properties of aqueous ethanol extracts of propolis on alpha-glucosidase.

Antibiofilm and Antioxidant Activity of Propolis and Bud Poplar Resins versus Pseudomonas aeruginosa.

Anti-Adipogenic Effects of Ethanol Extracts Prepared from Selected Medicinal Herbs in 3T3-L1 Cells.

Parasite Killing of Leishmania (V) braziliensis by Standardized Propolis Extracts.

Characterization and antioxidant properties of six Algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Antioxidant compounds from ethanol extracts of bamboo (Neosinocalamus affinis) leaves.

Dichloromethane extracts of propolis protect cell from oxygen-glucose deprivation-induced oxidative stress via reducing apoptosis.

Anti-Oxidant and Anti-Adipogenic Effects of Ethanol Extracts from Wheat Germ and Wheat Germ Fermented with Aspergillus oryzae.

Ethanol-Extracted Brazilian Propolis Exerts Protective Effects on Tumorigenesis in Wistar Hannover Rats.

Effects of Hylomecon vernalis ethanol extracts on cell cycle and apoptosis of colon cancer cells.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

Antioxidant activities and oxidative stress inhibitory effects of ethanol extracts from Cornus officinalis on raw 264.7 cells.

Pharmacological and phytochemical evaluation of Ocimum sanctum root extracts for its antiinflammatory, analgesic and antipyretic activities.

Thin-layer chromatographic identification of Chinese propolis using chemometric fingerprinting.

Effects of an ethanol extract of Brazilian green propolis on human cytochrome P450 enzyme activities in vitro.

Effects of Saccharomyces cerevisiae cell wall extract and poplar propolis ethanol extract supplementation on growth performance, digestibility, blood profile, fecal microbiota and fecal noxious gas emissions in growing pigs.

Effects of propolis on warfarin efficacy.

Alternative preparation of propolis extracts: comparison of their composition and biological activities.

Anticonvulsant potential of ethanol extracts and their solvent partitioned fractions from Flemingia strobilifera root.

Contact dermatitis from propolis.

Activity antifungal of the essential oils; aqueous and ethanol extracts from Citrus aurantium L.