Evaluation of anti-inflammatory effects of Broussonetia papyrifera stem bark.

OBJECTIVE: Broussonetia papyrifera is used as a traditional medicine to treat few diseases. However, the antiinflammatory effect of B. papyrifera stem bark has not been evaluated. The aim of this study is to investigate the effects of n-hexane fraction from methanol extract of B. papyrifera stem bark on lipopolysaccharide (LPS)-stimulated inflammation using RAW 264.7 cells.
MATERIALS AND METHODS: Methanol extract was obtained from B. papyrifera stem bark and its sequential fractions (hexane, dichloromathane, ethyl acetate, butanol, and aqueous) were obtained by extraction in solvents with increasing polarity and were examined in RAW 264.7 cells.
RESULTS: The secretion profiles of pro-inflammatory parameters, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were found to be significantly reduced in 10-80 μg/ml dose ranges of n-hexane fraction (BP-H) from methanol extract of B. papyrifera stem bark. The expressions of inducible NO synthase (iNOS) was also significantly inhibited by BP-H. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that BP-H treatment decreased LPS-induced iNOS mRNA expression in RAW 264.7 cells.
CONCLUSION: The results suggest that the B. papyrifera stem bark has anti-inflammatory activity which inhibits the NO production and proinflammatory cytokines in RAW 264.7 cells. B. papyrifera stem bark might act as a potential therapeutic agent for inflammatory diseases.

Introduction

Lipopolysaccharide (LPS) is a gram-negative bacterial endotoxin that could stimulate macrophage and monocytes through the binding of the LPS-binding protein (LBP) to CD14, a membrane glycophosphatidylinositol anchores protein. RAW 264.7, a murine macrophage-like cell lines, often play an important role in secreting inflammatory mediators and especially associate with endotoxin effects.[1] Pro-inflammatory genes are activated via signal transduction pathways that lead to the production of proinflammatory parameters such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β).[2-4] NO is produced by activating inflammatory cells. NO is synthesized from L-arginine through the action of constitutively expressed NO synthase (eNOS) and an inducible NO synthase (iNOS).[56] The augmentation of NO production by LPS is solely dependent on the expression of iNOS. Once iNOS is induced, it produces excessive amounts of NO which profoundly influence cell and tissue function and damage.[7] In the biological system, NO has many functions such as vasodilation, neurotransmission, and inflammatory response.[8] TNF-α is produced in the early phase of inflammatory cells, such as macrophages. TNF-α mediates early-stage responses of inflammation by regulating the production of other cytokines, including IL-1β and IL-6. Therefore, it is suggested as an important therapeutic approach to regulate the production of proinflammatory mediators for the treatment and prevention of inflammatory diseases.

The Broussonetia papyrifera is a tree in the family Moraceae, which grows well in Eastern Asia and also widely found in Taiwan. This plant has been used as traditional medicine for diuresis, homestasis, and for the relief of edema and cough.[910] Many polyphenolic compounds are known to possess antioxidant, anti-inflammatory, and anti-tumor activities.[1112] Previous studies have revealed that phenolic compounds, flavonoids, and alkaloids isolated from the leaves, fruits, and root of B. papyrifera have significantly shown the natural biological activities.[1314] However, the B. papyrifera stem bark is generally used for making high-quality papers or clothes, the anti-inflammatory effect of B. papyrifera stem bark has not been cleared.

The aim of this study is to investigate the influence of B. papyrifera stem bark on LPS-stimulated inflammation in vitro. The study investigated NO, TNF-α, IL-1β, and the expression of iNOS in RAW cells to evaluate the anti-inflammatory effects.

Materials and Methods

Chemicals and Reagents

Dulbecco's modified Eagle's medium, FBS (fetal bovine serum), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY, U.S.A.). Horseradish peroxidase-coupled anti-mouse and anti-rabbit antibodies and the ECLR detection reagent were purchased from Amersham Biosciences (Piscataway, NJ, U.S.A.). Antibodies for β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti- iNOS was purchased from BD Biosciences (Frankin lakes, NJ, U.S.A.). LPS was obtained from Sigma (St. Louis, MO, U.S.A.). All the other chemicals used including the solvents were of analytical grade. All materials for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.).

Preparation of Extracts

B. papyrifera was collected from Pingtung in June-September 2009. The dried stem bark of B. papyrifera was extracted with methanol for a week. The extract was filtered and removed the solvents removed under reduced pressure in a rotary evaporator to yield dried crude total extracts. The crude extract was dissolved in methanol: water (1:9) and subjected to sequential extraction with hexane, dichloromethane, ethyl acetate, and butanol. Each fraction thus obtained, including the final aqueous fraction, was evaporated under reduced pressure. In order to identify the different chemical ingredients, the extract was submitted to phytochemical procedures.[13] For in vitro experiments, the dried extract was dissolved to 100 mg/ml with DMSO and stored at –20°C until use.

Cell Culture

Murine RAW264.7 macrophages obtained from A.T.C.C. (Manassas, VA, U.S.A.) were grown at 37°C in 5% CO2 using Dulbecco's modified Eagle's medium containing 10%(v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. When examining the effects of drug, cells were treated with vehicle (DMSO) as a control.

Measurement of Cell Viability

Cell viability was assessed by the MTT assay.[15] To determine if agents treatment was cytotoxic, RAW 264.7 mouse macrophage cells plated in 24-well plates were treated with BP-H concentrations (10–80 μg/ml). After 24 h of incubation, MTT (1 mg/ml) was added for 3 hr, then the culture medium was removed, and the cells were dissolved in DMSO and shaken for 10 min. OD values at 550 and 630 nm were measured using a microplate reader. The net absorbance (OD550-OD630) indicates the enzymatic activity of mitochondria and provides information on cell viability.

Nitrite Measurement

Nitric oxide secreted by RAW 264.7 cells were measured by Griess reagent.[16] RAW cells were cultured in 24-well plates in 500 μl of culture medium until confluence. They were treated with LPS in the absence or presence of agents for 24 h and then the culture media were collected. Nitrite was measured by adding 100 μl of Griess reagent (1% sulphanilamide and 0.1% naphthylethylenediamide in 5% (v/v) phosphoric acid) to 100 μl samples of the culture medium. The absorbance at 550 nm (OD550) was measured using a microplate reader and the nitrite concentration was calculated by comparing with the OD550 produced using standard solutions of sodium nitrite in the culture medium.

Enzyme-linked Immunosorbent Assay (ELISA)

RAW264.7 cells cultured in 24-well plate were stimulated with indicated agents and cultured for 24 h. TNF-α and IL-1β productions were, respectively, measured by commercial kit (Cayman Chemical Company) according to the manufacturer's instruction.

Immunoblotting Analysis

After stimulation, cells were rinsed twice with ice-cold PBS and 100 μl of cell lysis buffer (20 mM Tris/HCl, pH 7.5/125 mM NaCl/1% Triton X-100/1 mM MgCl2/25 mM β-glycerophosphate/ 50 mM NaF/100 μM Na3VO4/1 mM PMSF/10 μg/ml leupeptin/10 μg/ml aprotinin) was then added to each plate. Protein was denatured in SDS, electrophoresed on SDS/polyacrylamide (10% gel), and transferred on to nitrocellulose membrane. Non-specific binding was blocked with TBST (50 mM Tris/HCl, pH 7.5/150 mM NaCl/0.1% Tween 20) containing 5% (w/v) non-fat milk for 1 h at room temperature. After incubation with the appropriate first antibodies, membranes were washed three times with TBST. The secondary antibody was incubated for 1 h. After three washes with tris-buffered saline and Tween 20 (TBST), the protein bands were detected with the ECL® reagent.

RT (Reverse Transcription) –PCR

To amplify iNOS mRNA, the specific primers for RT-PCR analysis were synthesized. Macrophages treated with the indicated agents were homogenized with 1 ml of RNAzol B reagent (Gibco) and total RNA was extracted by acid guanidinium thiocyanate/phenol/chloroform extraction. RT was performed using StrataScript RT–PCR kit and 10 mg of total RNA was reverse transcribed to complimentary DNA (cDNA) according to the manufacturer's instructions.[17] RT-generated cDNA encoding iNOS and β-actin genes were amplified using PCR. The oligonucleotide primers used correspond to the mouse iNOS (5’-CCC TTC CGA AGT TTC TGG CAG CAG C-3’ and 5’-GGC TGT CAG AGC CTC GTG GCT TTG G-3’), and mouse β-actin (5’- GACTACCTCATGAAGATCCT-3’ and 5’-CCACATCTGCTGGAAGGTGG-3’). PCR was performed in a final volume of 50 μl containing Thermus aquaticus (Taq) DNA polymerase buffer, all four dNTPs, oligonucleotide primers, Taq DNA polymerase, and RT products. After an initial denaturation for 2 min at 94°C, 35 cycles of amplification (94°C for 1 min, 58°C for 30s and 72 °C for 30s) were performed followed by a 10 min extension at 72°C. PCR products were analyzed on 2% (w/v) agarose gel. The mRNA of β-actin served as an internal control for sample loading and mRNA integrity.

Statistical Evaluation

Values are expressed as mean ± S.E.M. for at least three experiments which were performed in duplicate. Analysis of variance (ANOVA) was used to assess the statistical significance of the differences, and a P<0.05 was considered to be statistically significant.

Results

Anti-inflammation of Fractions From Broussonetia papyrifera

With increasing solvent polarity, hexane (H), dichloromethane (Di), ethyl acetate (Ac), butanol (B), and aqueous (Aq) fractions from the methanol extract of B. papyrifera stem bark were evaluated on LPS-induced inflammation in RAW 264.7 cells. As shown in Figure 1, hexane fraction, dichloromethane fraction, and ethyl acetate fraction could inhibit significantly the LPS-induced NO production. Among these fractions, hexane fraction obtained a maximum inhibition of the inflammatory activity. Therefore, hexane fraction (BP-H) of B. papyrifera stem bark was determined for further studies.

Figure 1

Effect of hexane (H), dichloromethane (Di), ethyl acetate (Ac), butanol (B), and aqueous (Aq) fractions from methanol extract of B. papyrifera stem bark on LPS-induced NO production in RAW 264.7 cells. Cells were treated with various 100 μg/ml of BP fractions indicated for 30 min followed by the stimulation with LPS (100 ng/ml) for 24 hr. The data are the mean ± S.E.M. from at least three independent experiments, each performed in duplicate. The asterisks denote the significance levels when compared to control groups. Significantly different from controls, *P<0.05 and **P<0.01

Effect of LPS-Induced NO Production and iNOS Protein

To study the effect of BP-H on LPS-induced NO production and iNOS protein in RAW264.7 cells, RAW cells were treated with 10, 30, 50 and 80 μg/ml of BP-H and LPS (100 ng/ml) for 24 hr. After incubation, the culture medium was collected for nitrite assay and cell lysates were subjected to SDS-PAGE for iNOS measurement. Co-addition of BP-H with LPS inhibited the formation of NO in a concentration-dependent manner and the estimated mean IC50 value of BP-H was 32.15 μg/ml. Expression of iNOS protein was also decreased in the presence of BP-H in a concentration-dependent manner as well [Figure 2].

Figure 2

Effect of BP-H on NO production and iNOS protein in LPS-induced RAW 264.7 cells. RAW cells were treated with 10, 30, 50 and 80 μg/ml of BP-H and LPS (100 ng/ml) for 24 hr. After incubation, the culture medium was collected for nitrite assay and cell lysates were subjected to SDS-PAGE for iNOS measurement. Changes in iNOS protein levels normalized by β-actin were quantified. The data are the mean ± S.E.M. from at least three independent experiments, each performed in duplicate. The asterisks denote the significance levels when compared to control groups (* P<0.05)

Levels of TNF-α and IL-1β in Medium

In the LPS group, the TNF-α concentration was 1100 ± 42.5 pg/ml. Coaddition of BP-H with LPS showed a tendency to decrease the TNF-α concentration in a dose-dependent manner. Treatment with BP-H (10-80 μg/ml) significantly reduced TNF-α concentration compared to that of the LPS group (10 μg/ml: 971.8 ± 38.9 pg/ml; 30 μg/ml: 693.9 ± 50.5 pg/ml; 50 μg/ml: 342.3 ± 67.3 pg/ml; 80 μg/ml: 163.6 ± 43.1 pg/ml) [Figure 3]. In addition, the IL-1β concentration in the BP-H group also showed a tendency to decrease in dose-dependent manner. In the LPS group, the IL-1β concentration was 95.2 ± 12.1 pg/ml. Treatment with BP-H (10-80 μg/ml) significantly reduced IL-1β concentration compared to that of the LPS group (10 μg/ml: 88.3 ± 6.5 pg/ml; 30 μg/ml: 65.3 ± 10.2 pg/ml; 50 μg/ml: 41.2 ± 7.2 pg/ml; 80 μg/ml: 18.4 ± 3.5 pg/ml) [Figure 4].

Figure 3

Effect of BP-H on TNF-α in LPS-induced RAW 264.7 cells. RAW cells were treated with 10, 30, 50, and 80 μg/ml of BP-H and LPS (100 ng/ml) for 24 hr. The data are the mean ± S.E.M. from at least three independent experiments, each performed in duplicate. The asterisks denote the significance levels when compared to control groups (*P<0.05)

Figure 4

Effect of BP-H on IL-1β in LPS-induced RAW 264.7 cells. RAW cells were treated with 10, 30, 50, and 80 μg/ml of BP-H and LPS (100 ng/ml) for 24 hr. The data are the mean ± S.E.M. from at least three independent experiments, each performed in duplicate. The asterisks denote the significance levels when compared to control groups (*P<0.05)

MTT Assay for Cell Viability

RAW cells were treated with BP-H concentrations (10-80 μg/ml) for 24 h and cell viability was examined by using MTT assay. The result indicated that the highest concentration of BP-H did not cause the cell death. BP-H showed no cytotoxic effect on RAW cells compared to the control cells [Figure 5].

Figure 5

Effect of BP-H on cell viability. RAW 264.7 cells were treated with 10, 30, 50, and 80 μg/ml of BP-H at the concentrations indicated for 24 hr and cell viability assay was performed using MTT dye as mentioned in materials and methods

Inhibition of mRNA Expression of iNOS

To understand further whether iNOS protein inhibition was derived from gene transcription or not, RT–PCR analysis of mRNA was performed. As shown in Figure 6, RAW cells were treated with BP-H concentrations (10-80 μg/ml), which revealed a gradual decreased level of iNOS mRNA. The results indicated that the decreased mRNA expression was associated with BP-H in a dose dependent manner. However, the expression of iNOS gene regulated by BP-H has not been elucidated, the gene regulation need to be further examined.

Figure 6

Effect of BP-H reduced iNOS mRNA expression. Following the treatment with BP-H as indicated for 12 hr, changes in iNOS mRNA levels were measured by the PCR products. RNA isolation and the RT-PCR process were carried out as described. The β-actin mRNA level was considered the internal control

Discussion

The medical plant, B. papyrifera, exhibited many pharmaceutical responses, such as antinociceptive, antioxidant, antimicrobial, and anti-inflammatory activities.[18] About 42 compounds were isolated from the whole plants of B. papyrifera.[19] Phenolic compounds with antioxidant activity were isolated from the fruits of B. papyrifera against H2O2-induced neuronal injury in human neuroblastoma SH-SY5Y cells[2021] or against H2O2-induced impairment in PC12 cells.[22] Some broussonetones-like compounds isolated from the leaves of B. papyrifera have shown antioxidant and antityrosinase activities and they could be useful ingredients in the development of skin-protecting cosmetics.[23] The organic extract of the roots of Broussonetia papyrifera showed extremely high alpha-glucosidase inhibitory activity to identify their inhibitory potencies and kinetic behavior.[24] In this study, phytochemical analysis revealed the presence of tannins, saponins, flavonoids, alkaloids, and reducing sugars in the methanol extract B. papyrifera stem bark, while the n-hexane fractions showed the presence of flavonols and chalcone derivatives as major compounds.

Previous studies have reported the anti-inflammatory properties of plant extract on LPS-induced inflammation. Dongmo et al.[25] reported that the stem bark extracts of Mitragyna ciliata possess potent anti-inflammatory effects. Akah et al.[26] evaluated that the leaf extracts of Asystasia gangetical exhibited anti-inflammatory and anti-asthmatic activity. Goming et al.[27] studied the anti-inflammatory effect of Serjania erecta on croton-oil-induced inflammation in mouse models. In addition, Deena and Thoppil[28] tested the antimicrobial activity of the essential oil of Lantana camara against bacteria and fungi.

In the present investigation, we examined the anti-inflammatory properties of hexane, dichloromethane, ethyl acetate, butanol, and aqueous fractions from methanol extract of B. papyrifera stem bark on LPS-induced NO production in RAW cells. The results demonstrated that administration of methanol extract from B. papyrifera was capable of inhibiting the inflammation response induced by LPS, namely bacterial endotoxin. The fractionation of B. papyrifera extract produced with hexane, dichloromethane, and ethyl acetate generated fractions that could inhibit NO production caused by LPS. In contrast, the aqueous and butanol fractions were ineffective in reducing NO release. This can be explained by the high polarity of the solvents used in these fractions, which extract polar compounds that are either poorly absorbed through the cell or lack anti-inflammatory activity.

In this study, we have observed that the hexane fraction of B. papyrifera showed the strongest anti-inflammatory activity (ID50 = 32.15 μg/ml), exhibited greater potency than dichloromethane and ethyl acetate fractions. The extracts exhibited anti-inflammatory activity in the order of magnitude – hexane extract > ethyl acetate > dichloromethane extract. In fact, it was the only one with a higher potency than the methanol extract. Therefore, we thought that most of the anti-inflammatory compounds or the compounds with the highest potency were concentrated in the hexane fraction.

In conclusion, the results revealed the anti-inflammatory activity of B. papyrifera stem bark on LPS-induced proinflammatory parameters in RAW 264.7 cells. The NO release, iNOS protein, and mRNA expression was decreased by BP-H. However, the expression of iNOS gene regulated by BP-H has not been elucidated and that the gene regulation need to be further examined.