The Ethanol Extract of Osmanthus fragrans Flowers Reduces Oxidative Stress and Allergic Airway Inflammation in an Animal Model

The Osmanthus fragrans flower, a popular herb in Eastern countries, contains several antioxidant compounds. Ben Cao Gang Mu, traditional Chinese medical literature, describes the usefulness of these flowers for phlegm and stasis reduction, arrest of dysentery with blood in the bowel, and stomachache and diarrhea treatment. However, modern evidence regarding the therapeutic efficacy of these flowers is limited. This study was aimed at assessing the antioxidative effects of the ethanol extract of O. fragrans flowers (OFE) in vivo and evaluating its antioxidant maintenance and therapeutic effect on an allergic airway inflammation in mice. After OFE's oral administration to mice, the values obtained in the oxygen radical absorbance capacity assay as well as the glutathione concentration in the lungs and spleens of mice increased while thiobarbituric acid reactive substances decreased significantly, indicating OFE's significant in vivo antioxidant activity. OFE was also therapeutically efficacious in a mouse model of ovalbumin-induced allergic airway inflammation. Orally administered OFE suppressed ovalbumin-specific IgE production and inflammatory cell infiltration in the lung. Moreover, the antioxidative state of the mice improved. Thus, our findings confirm the ability of the O. fragrans flowers to reduce phlegm and suggest that OFE may be useful as an antiallergic agent.

asthma for total [22] and for oxidized [23] GSH. Total GSH concentration, which includes the oxidized form GSSG, is higher in the bronchial and alveolar fluid in patients with mild asthma [22]. A recent report suggested that increasing GSH levels and the GSH/GSSG ratio with -glutamylcysteine ethyl ester ( -GCE) ameliorated bronchial asthma by altering the Th1/Th2 cell imbalance through interleukin (IL-) 12 production from antigen presenting cells (APC) and by suppressing chemokine production and eosinophil migration [24]. Enhancing intracellular GSH can also decrease the release of cytokines from lung cells by decreasing NF-B activation [25,26]. In addition, GSH has recently been reported to attenuate IL-13 induced asthma in mice [27].
Liao et al. investigated the total antioxidant capability in serum and the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase in 46 asthmatic children and 52 normal controls [28]. The total antioxidant capability in the serum of asthmatic children is significantly less than that found in the serum of controls. Plasma levels of lipid peroxides are also significantly increased in patients with asthma [29,30].
The concentration of ascorbic acid and -tocopherol in lung lining fluid is reportedly low in patients with mild asthma [23,31]. Low dietary intake of vitamin C and manganese was found to be associated with an increased risk of bronchial activity [30]. Dietary supplements of vitamin C and vitamin E to asthmatics decrease the severity of pollutant-induced bronchial responsiveness [32]. Chang and Crapo [33] found that intratracheal treatment of ovalbumin (OVA-) challenged mice with the catalytic antioxidant, manganese (III) meso-tetrakis-(N-methylpyridinium-2-yl) porphyrin, having high SOD activity drastically reduced the severity of airway inflammation as evidenced by the reduced numbers of eosinophils, neutrophils, and lymphocytes found in bronchoalveolar lavage fluid.
The aim of the current study was to examine the mechanism of the preventive effect of OFE on allergic airway inflammation. The study comprised two parts: the first was to evaluate the antioxidation state following OFE administration and the second was to examine the immunomodulatory effects of OFE. We evaluated the antioxidative effects of OFE in naïve mice using the following methods: assessing the total antioxidant capacity in the lung and spleen by using the values obtained in the oxygen radical absorbance capacity (ORAC) assay, assessing the free radical-scavenging effects on DPPH, monitoring lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS), and determining the concentration of GSH. We used a mouse model of allergic airway inflammation to investigate the antiallergic and antioxidant effects of OFE as well as to determine organ weights.

Preparation of OFE. The dried flowers of O. fragrans
were ground into a fine powder using a mill (RT-08, Rong Tsong, Taiwan), collected and sealed in a polyethylene plastic bag, and then stored at 0-4 ∘ C for further use. O. fragrans flowers (200 g) were soaked (72 h) in 75% ethanol (3 L) twice and filtered through Whatman no. 1 filter paper. The combined extracts were concentrated under reduced pressure and freeze-dried to provide a dark syrup that was stored at −20 ∘ C for further use. The extract was endotoxin free (≤0.1 E.U.) according to the Limulus amebocyte lysate (LAL) assay (Cambrex, Walkersville, MD).

Determination of Total Phenolic Content in OFE.
Following the method described by Yen and Hung [34], the sample solution in methanol (0.1 mL, 1 mg/mL) was well mixed with 2% Na 2 CO 3 (2 mL). After 3 min, 50% Folin-Ciocalteu agent (0.1 mL) was added. The mixture was allowed to stand at room temperature (RT) for 30 min with intermittent mixing. The absorbance at 750 nm was recorded. A standard curve using gallic acid was prepared. The total phenolic content was expressed as gallic acid equivalents (mg of GAE per g extract).

Determination of Total Flavonoid Content in OFE.
Following the methods described by Woisky and Salatino [35] and also by Chang et al. [36], the sample solution (0.5 mL) was mixed with 95% EtOH (1.5 mL), 10% AlCl 3 (0.1 mL), 1 M KOAc (0.1 mL), and distilled water (2.8 mL). The mixture was allowed to stand at RT for 30 min, and the absorbance was measured at 415 nm. The amount of sample solution was substituted by the same amount of a quercetin solution (0-200 g/mL) as a standard. The amount of 10% aluminum chloride was substituted by the same amount of distilled water to serve as a blank. The total flavonoid content was calculated from the plot of absorbance against quercetin concentration using linear regression analysis and expressed as quercetin equivalents ( g of QE per g extract).

DPPH Free Radical-Scavenging
Assay. DPPH is a stable free radical with a purple color that is reduced by antioxidants to a colorless compound. We employed DPPH in an assay modified from the method of Shimada et al. [37]. MeOH (3.8 mL), sample solution in methanol (0.2 mL, 1 mg/mL), and 1 mM DPPH solution (1.0 mL) were mixed well and left to stand in the dark at RT for 30 min. The final concentration of the sample was 40 g/mL. The absorbance at 517 nm was measured. The sample in methanol was used as a blank, while DPPH radical in methanol solution was used as a control. The DPPH radical scavenging activity was calculated according to the following equation: % of DPPH radical scavenging activity where is the absorbance at 517 nm.
The concentration providing 50% inhibition (IC 50 ) of the DPPH radical-scavenging activity was calculated from the plot of the percent inhibition against sample concentration by using a linear regression analysis.

Animals.
Female BALB/c mice between six and eight weeks of age were purchased from the National Laboratory Animal Center in Taiwan. The animal room was kept on a 12 h light : dark cycle and maintained at a constant temperature (25 ∘ C ± 2 ∘ C) and humidity. Animal care and handling conformed to the NIH Guide for the Care and Use of Laboratory Animals. All experiments were performed under protocols approved by the biotechnology department of National Formosa University's affidavit of approval of animal use protocol. Pentobarbital (intraperitoneal, i.p.) injections were used to anesthetize (10 mg/mL, 60 L per mouse) or sacrifice (10 mg/mL, 200 L per mouse) the mice.
For the results obtained in the antioxidant evaluation assays, mice were divided into two groups comprised of four BALB/c mice each. Group 1 (control) received only distilled water. Group 2 (OFE) received 1000 mg/kg body weight OFE daily for 14 days by oral gavage.

Organ Collection, Preparation, and Protein Quantization.
After sacrifice, organs from the mice were collected and weighed. The spleens and lungs (1 g) were homogenized in PBS (pH 7.2) on ice by using a homogenizer (Motor Drives, Glas-Col Inc., Terre Haute, IN). The homogenate was then centrifuged at 2200 ×g for 10 min, and the filtrate was collected. Protein was determined by using the BCA assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Oxygen Radical Absorbance Capacity Assay (ORAC).
The total antioxidant activity of the organ samples was measured by using the oxygen radical absorbance capacity (ORAC) assay according to Chung et al. [38]. This assay was carried out in black-walled, 96-well plates at 37 ∘ C. All solutions were prepared in 75 mM phosphate buffer (Na 2 HPO 4 : NaH 2 PO 4 , pH 7.0) and preincubated at 37 ∘ C for 30 min before use. Fifteen L of organ homogenate (diluted 100 times) and 100 L of 0.1 M -PE ( -phycoerythrin) were transferred directly into the well to incubate for 10 min using the FLUOstar OPTIMA microplate reader system (Galaxy BMG LABTECH Inc., Cary, NC). We rapidly added 75 mM 2,2 -azobis (2-amidinopropane) dihydrochloride (AAPH, 85 L) and immediately measured the resulting fluorescence by using fluorescence filters with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The fluorescence was recorded at 5 min intervals for 120 min until the final value was less than 5% of the initial value. ORAC values from samples were calculated by using the following equation and expressed as Trolox equivalents: ORAC value Evidence-Based Complementary and Alternative Medicine the sample dilution factor. The area under the curve ( ) was calculated by the following equation: where 0 was the initial fluorescence reading at 0 min and represented the measurement at time .

TBARS Assay.
Lipid peroxidation was measured by determining the formation of malondialdehyde based on the presence of TBARS in the lung and spleen [39]. A standard curve was prepared using 1,1,3,3-tetraethoxypropane (Sigma, St. Louis, Mo). The organ filtrate (1 mL) and standard were mixed with 10% trichloroacetic acid (1 mL) 0.4% thiobarbituric acid (1 mL) and 0.2% butylated hydroxy toluene (BHT) (0.1 mL) (both reagents from Sigma, St. Louis, Mo). The reaction mixture was incubated at 95 ∘ C for 1 h, cooled under light-protected conditions, and then centrifuged at 2200 ×g for 10 min. The absorbance of the supernatant was measured using a microplate fluorescence reader (F-2500, Hitachi, Japan) in 96-well format with the excitation and emission filters set at 515 nm and 550 nm, respectively.

Measurement of Glutathione.
The glutathione concentration was determined according to the method described by Sedlak and Lindsay [40] with little modification. The organ homogenate (150 L) was mixed with 5% TCA solution (450 L). The resulting solution was centrifuged at 16770 ×g for 10 min to remove protein. The supernatant (30 L) and 0.01 M 5,5 -dithio-bis-(2-nitrobenzoic acid) (DTNB, 10 L) were added to 0.4 M Tris buffer (140 L, pH 8.9). The solution remained at RT for 5 min. The absorbance at 412 nm was measured by n ELISA Reader (VersaMax, Molecular Devices Inc., Sunnyvale, CA). A GSH solution (0-12 M) was used as standard. Tris buffer was used as blank. The plasma GSH content was calculated from the plot of absorbance against GSH concentration.

Establishment of the Animal Model of Allergic Airway
Inflammation. The schedule for OVA sensitization and challenge as well as the oral administration schedule of OFE are summarized in Figure 1. Briefly, the mice were sensitized with i.p. injections of OVA (20 g in 1x PBS; Sigma) mixed with alum (2 mg; Pierce, Rockford, III) as an adjuvant on day 0. On day 14, the mice were boosted with OVA (50 g) mixed with alum (2 mg). The mice that were given i.p. injections of 1x PBS served as negative controls. Either one of two doses of OFE in normal saline (200 L) (high OFE, 1000 mg/kg body weight or low OFE, 100 mg/kg body weight) or normal saline (for positive and negative control groups) was orally administered daily from day 1 to day 29. Each group consisted of six mice. On days 35-38, the mice were exposed to aerosolized 5% OVA for a 20 min period by placing them in a chamber. The aerosols were generated and conducted into the chamber using an ultrasonic nebulizer (DeVibiss, PA). The output of the nebulizer was 0.3 mL/min and the particles size produced was 0.5-5 m.

Serum Levels of Anti-OVA Antibodies.
After all groups of mice were anesthetized, they were bled from the retroorbital venous plexus. Serum was collected after centrifugation at 12,000 ×g for 10 min. Sera anti-OVA IgE and IgG2a antibody titers were determined by ELISA. Briefly, 96-well microtiter plates were coated with OVA (10 g/mL). Serum samples were added to each well for an overnight period. Then biotin-conjugated anti-mouse IgE or IgG2a was added for 1 h at 37 ∘ C. Streptavidin-conjugated alkaline phosphatase was added for an additional 2 h at RT. Finally, the reaction was developed by 2,2 -azino-bis (3-ethylbenz-thiazoline-6sulfonic acid), and the absorbance was determined at 420 nm in a microplate reader.

Bronchoalveolar Lavage and Cell Differential Counts.
After all groups of mice were anesthetized, they were bled from the retroorbital venous plexus and then sacrificed. Using a cannula, their lungs were immediately lavaged through the trachea three times with 1x HBSS (1 mL) minus ionized calcium and magnesium. The lavaged fluid was centrifuged at 400 ×g for 10 min at 4 ∘ C. After washing, the cells were resuspended in 1x HBSS (1 mL) and the total cell counts were determined using a hemocytometer. Cytocentrifuged preparations were stained with Liu's stain for differential cell counts. Based on standard morphologic criteria, a minimum of 200 cells was counted and classified as monocytes, lymphocytes, neutrophils, or eosinophils.

Histopathological Study of Lung Organs.
To evaluate the effects of OFE treatment, the lungs were immediately removed after lavage and fixed in a solution of 3% v/v formalin (in 0.01 M phosphate buffer, pH 7.2). The tissues were subsequently embedded in paraffin, cut into 5-m-thick sections, stained with hematoxylin-eosin, and examined by light microscopy for histopathological changes.

Statistical
Analysis. Data from the ORAC and the TBARS assay as well the GSH concentration and various treatments are presented as mean ± SD (standard deviation). The Student's t test was used for comparison between treatments. Differences between two treatment groups or a treatment group compared with a negative or positive control group were considered statistically significant at values less than 0.05, 0.01, or 0.001.

The Total Phenolic and Flavonoid Content in OFE.
The total phenolic content in OFE was 367.9 ± 13.4 mg GAE/g extract, while the total flavonoid content was 45.0 ± 2.0 g QE/g extract. Flavonoids have been reported to induce antiallergic and anti-inflammatory effects [41]. For example, flavonoids showed a strong inhibition of IL-4 and IL-13 production, histamine release, and CD40 ligand expression in Evidence-Based Complementary and Alternative Medicine 5  basophils [42]. The antiasthmatic effect by phenylpropanoid glycosides such as verbascoside has also been reported [43].

Antioxidant Activity and Free Radical Scavenging Capacity of OFE as Determined in the ORAC and DPPH Assays.
The total antioxidant capacity of ethanol extract of OFE as determined in the ORAC assay was 0.4 ± 0.0 mM Trolox equivalents. The DPPH IC 50 was 8.4 g/mL, which was better than that of the methanol extract (12.8 g/mL) of O. fragrans flowers [3], but less than Trolox (4.9 g/mL). Green tea, which has been proven to have a strong scavenging effect against DPPH radicals, has been reported to have antiallergic activity [44]. We found that in addition to the other antioxidant phenolic compounds, verbascoside and rutin are two major compounds in the ethanol extract of OFE (unpublished data). The antiallergic effects of verbascoside [43] and rutin [45] have also been reported. Together, these results support our decision to evaluate the antiallergic effects of the ethanol extract of OFE. damage of biological molecules. Additionally, oxidation is believed to play a role in the development of inflammatory diseases. For this reason, the total antioxidant capacity (as measured in the ORAC assay), GSH concentration, and lipid peroxidation (as measured in the TBARS assay) in mouse lung and spleen were investigated. There are no previous reports on the in vivo antioxidant effects of OFE. We first evaluated the antioxidant capacity of the OFE extract in mice after oral administration of OFE for 14 days. Lungs and spleens from the OFE group demonstrated significantly higher values in the ORAC assay and higher concentrations of GSH than those in the control group (normal saline) ( Table 1). In contrast, values derived from the TBARS assay were significantly decreased in the OFE group compared with those in the control group. Taken together, these results indicate that oral administration of OFE promotes antioxidant capacity and reduces lipid peroxidation in the lung and spleen of mice.

The Effects of Orally Administered OFE on Total Antioxidant Capacity, Lipid Peroxidation, and GSH Concentration in Lung and
The phenolic antioxidants in OFE include tyrosyl acetate, (+)-phillygenin, (8E)-ligustroside, rutin, and verbascoside [3]. The verbascoside obtained from OFE has been reported to protect cell lines from free radical-induced oxidative stress as measured by TBARS [46] and -amyloid-induced cell injury by attenuating ROS production [46]. However, the in vivo evaluation of the antioxidant effects of these five compounds needs further study. Oxidative stress is an important factor that contributes to the pathologic development of asthma. Given our results for the antioxidant capacity of OFE, we next evaluated OFE for its preventive effects in an animal model of allergic airway inflammation.

OVA-Specific Serum Antibody Levels in an Animal
Model of Allergic Airway Inflammation. Following the OFE preventive protocol (Figure 1), OFE oral administration inhibited OVA-specific IgE production (Figure 2(a)) and enhanced the production of OVA-specific IgG2a (Figure 2(b)). IgE suppression can alleviate the development of asthma [47]. In contrast, IgG2a is the major isotype of the Th1 immune response. The effect of antioxidants to regulate the immune response has been an important issue in western society [48]. The strong Th2 inhibition by flavonoids [42] and phenolic compounds [49] contributes to the suppression of Th2 inflammation. The polarization effect of antioxidants  on Th1 and Th2 has been discussed [50]. In a study of the antiallergic effects of curcumin, IFN-was increased and IgE was suppressed in the latex allergy model [51]. In our study, the increase in anti-OVA IgG2a and decrease in IgE expression indicate that OFE prevents development of Th2 suppression. A higher GSH induction also contributes to the development of Th1 as suggested by the Th1-promoting effect of GSH [24]. demonstrated that these compounds in OFE contribute to the inhibition of inflammatory cell infiltration in lung.

Effects of Oral OFE Administration on Relative Organ Weight in an Animal Model of Allergic Airway Inflammation.
A change in organ weights is a good indicator of chemically-or biologically-induced damage to organs [52]. Oral administration of OFE (100, 800, or 1000 mg/kg) for 28 days did not alter organ weights (liver, spleen, lung, and kidney) in mice (data not shown). However, HOFE (1000 mg/kg) administration for 28 days to mice subjected to the OVA animal model of allergic airway inflammation lowered the relative kidney weight to body weight percentage when compared with mice in the negative control group (NC group, treated with saline) but not when compared with mice in the positive control (PC group, OVA-immunized and treated with saline) ( Table 2). In contrast, there were no significant differences among groups in the relative organ weight to body weight percentages for liver and lung. Recently, we evaluated a medium dose of OFE (500 mg/kg) in the OVA-induced allergic airway inflammation model and found that medium dose of OFE also protected against the infiltrated cells in BAL fluid and maintained the antioxidative state in lung (data not shown). Further investigation will be required to clarify the organ cytotoxicity effect by a medium dose of OFE orally administered in this animal model of allergic airway inflammation.

OFE Improves Antioxidative Status in Mouse Lung following OVA Administration.
We measured the severity of the oxidative damage in lungs of mice from each group. In lungs taken from animals subjected to the mouse model of allergic airway inflammation, the GSH concentration and the total antioxidant capacity values obtained from the ORAC assay were significantly decreased compared with those of the lungs from mice in the negative control group. This suggests that airway inflammation may lead to oxidative stress in lungs. However, these phenomena were improved following oral administration of OFE (Figures 5(a) and 5(b)). In contrast, the value obtained in the TBARS assay for mice treated with OFE was significantly lower than that obtained for mice in the positive control group ( < 0.01, Figure 5(c)). Taken together, these data indicate that OFE improves the total antioxidant capacity in lung and that this improvement is correlated with a lower severity of signs in the OFE-treated mice subjected to the OVA-induced animal model of allergic airway inflammation. Recent reports have suggested that increasing the GSH levels could attenuate Th2 development [24][25][26][27], which offers a possible mechanism for the alleviation in the severity of airway inflammation observed in the present study following OFE administration.

Conclusion
The therapeutic success of many antioxidant agents in allergic airway inflammation in the clinic is moderate at best [53,54]. Conversely, researchers have found that antioxidants such as fruit juice [55] and plant extracts [56] interfere with the Th1 immune response in human peripheral blood mononuclear cells. Such results reflect the need for determining the appropriate antioxidants for treatment in different types of inflammatory disease. The present report was the first to evaluate the antioxidant and immunomodulatory effects of OFE, applying traditional Chinese medicinal knowledge regarding the O. fragrans flower to modern scientific study. We found that OFE, which contains many antioxidants, promotes a positive antioxidative state in an animal model of allergic airway inflammation. It also has protective effects including decreasing the OVA-specific IgE production and inflammatory cell infiltration in lung.