Phenolic Antioxidants Isolated from the Flowers of Osmanthus fragrans

O. fragrans has slightly less antioxidative activity than green tea. Five phenolic compounds, tyrosyl acetate (1), (+)-phillygenin (2), (8E)-ligustroside (3), rutin (4), and verbascoside (5), were isolated from the CHCl3 sub-extract of O. fragrans. The structures were elucidated by interpreting their spectral data. Evaluation of the antioxidative property of the isolated (+)-phillygenin (2), rutin (4), and verbascoside (5) revealed strong DPPH radical scavenging activity, with IC50 values of 19.1, 10.3, and 6.2 μM, respectively. These isolates also exhibited an H2O2 scavenging ability, with IC50 values of 10.5, 23.4, and 13.4 μM, respectively.


Introduction
In China, some flowers are commonly added to tea to increase or improve its taste. The added flowers are Gomphrena globosa, Helichrysum bracteatum, Chrysanthemum morifolium, Momordica grosvenori, Chrysanthemum indicum, Nelumbo nucifer, Osmanthus fragrans, and others. A preliminary antioxidative test was carried out to evaluate the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of a methanol extract of these flowers at a concentration of 40 μg/mL. The values obtained ranged from 3.4% to 91.3% (Figure 1a). O. fragrans exhibited the strongest activity OPEN ACCESS (91.3%), but a little less activity than green tea (93.4%), which has been proven to exhibit strong antioxidative activity [1,2]. Similarly, the total phenolic content of O. fragrans (291.3 mg/g extract) was determined to be a little less than that of green tea (325.9 mg/g extract), but it was the highest among the seven flowers tested (Figure 1b). Therefore, O. fragrans became the candidate for a constitution and pharmacological investigation. Each value is mean ± standard derivation of three replicates. Osmanthus is a genus of approximately 30 species of flowering plants of the family Oleaceae, mostly native to warm temperature regions of Asia ranging from the east of the Himalaya through southern China to Taiwan and southern Japan. The species Osmanthus fragrans Lour. is an evergreen, polygamous or dioecious shrub or small tree, which grows to 3-12 m tall. Its flowers are white, pale yellow, golden yellow, or orange yellow with a four-lobed corolla, and have a strong fragrance [3]. It is used not only as an ornamental plant, but also as an additive in food, tea, and other beverages because of its strong fragrance. Most components in the essential oil of O. fragrans have been identified by GC-MS [4,5]. Additionally, the flower of O. fragrans has been demonstrated to exhibit antioxidative activity [6], to inhibit NO production [7], to have a neuroprotective effect [8], and to (a) (b) inhibit melanogenesis [9,10]. It can be utilized as a herbal drug against toothache, as a gargle [11], or in cosmetic therapeutics [12]. However, the constituents responsible for the antioxidative effect are unknown. This study describes the isolation, structural elucidation of the purified compounds, and evaluates their antioxidative activity against DPPH radical and hydrogen peroxide (H 2 O 2 ).

Antioxidative Activity and Total Phenolic Content of Extracts
The antioxidative activity of the crude methanol extract was determined to be 91.3% at 40 μg/mL (IC 50 = 12.8 μg/mL) in the scavenging of DPPH radical and 72.4% at 24 μg/mL (IC 50 = 16.6 μg/mL) in the scavenging of H 2 O 2 ( Table 1). The total phenolic content was found to be 291.3 mg/g extract. Common natural antioxidants are phenolics, ascorbic acid, tocopherols, and carotenes. Ascorbic acid, tocopherols, and carotenes contents were not detected herein by the 2,6-dichloroindophenol (DIP) method or HPLC analysis [13]. Accordingly, the phenolic compounds were the dominant antioxidants in the methanol extract of O. fragrans.
The crude methanol extract of O. fragrans was suspended in H 2 O. The aqueous solution was partitioned with CHCl 3 to yield the CHCl 3 sub-extract. It exhibited strong antioxidative activity (87.4% at 40 μg/mL, IC 50 = 20.9 μg/mL). The phenolic components associated with the antioxidative activity of the CHCl 3 sub-extract were thus investigated.
From fraction 2, a white amorphous powder compound 1 was isolated. The pseudo molecular ion at an m/z of 181 [M+H] + in the FABMS had the molecular formula C 10 H 12 O 3 . The 1 H-NMR spectrum presented signals at δ 6.77 (2H, d, J = 8.0 Hz) and 7.07 (2H, d, J = 8.0 Hz) typical of a para-disubstituted benzene. A hydroxyl substituent on the ring was responsible for the broad signal at δ 5.29.   The other substituent was assumed to be an acetoxyethyl group associated with signals at δ 2.04 (3H, s), 2.86 (2H, t, J = 7.0 Hz), 4.24 (2H, t, J = 7.0 Hz). Therefore, compound 1 was determined to be p-(hydroxyphenyl)ethyl acetate, which is also called tyrosyl acetate [14].
From fraction 3, compound 2 was obtained as a white amorphous powder. The FABMS revealed I to be a molecule with a formula of C 21 H 24 O 6 , based on the presence of a pseudo molecular ion at an m/z of 373 [M+H] + . The UV and IR spectra suggested the presence of an aromatic ring. In the 1 H-NMR and COSY spectra, aromatic signals at δ 6.78 (1H), 6.83 (1H), 6.91 (2H) and 6.99 (2H) together with three methoxyl signals at δ 3.78, 3.80, and 3.83 and a hydroxyl signal at δ 7.58 revealed the presence of a 4'-hydroxy-3'-methoxyphenyl ring and a 3",4"-dimethoxyphenyl ring. The eight aliphatic signals at 2.89 (1H), 3.18 (1H), 3.39 (1H), 3.76 (2H) 4.10 (1H), 4.35 (1H), 4.83 (1H) represent two sets of mutually coupled CH 2 -CH-CH fragments that are linked by two oxygen atoms to form an unsymmetrical furofuran lignan. The cis-fused furofuran ring system was revealed by the strong NOE correlation between H-1 and H-5. The NOE correlation between H-1 and H-2, the absence of an NOE correlation between H-5 and H-6 and the positive specific rotation ([α] D = +88°) suggest that the absolute structure of 2 is that of (+)-phillygenin [15].
From fraction 6, a compound 5 was isolated as a white amorphous powder. Its molecular weight was 624, as determined by FABMS, consistent with the molecular formula C 29 H 36 O 15 . The UV and IR spectra showed the presence of hydroxyl and conjugated carbonyl functional groups. Like rutin (4), two sugars, a glucose and a rhamnose, were revealed by the anomeric signals at The key HMBC correlations of glucosyl H-1" with phenylethyl C-8; glucosyl H-4" with caffeoyl C-9', glucosyl H-3" with rhamnosyl C-1"' revealed that 5 had the verbascoside (acteoside) The spectral data of 5 agreed closely with those reported in the literature [18].
If we focus on the aromatic rings, the structures of phenolic compounds 1-5 differ mainly in the number and position of hydroxyl or methoxyl groups that are attached to the phenyl ring: tyrosyl acetate (1) has a tyrosyl group with only a p-phenolic hydroxyl group; (+)-phillygenin (2) contains two methylated catechol moieties-one with a methoxyl group and a free hydroxyl group, the other with two methoxyls; (8E)-ligustroside (3) has a tyrosyl unit such as 1; rutin (4) is a flavonoid with two hydroxyl groups in the B ring and a rutinose that is attached to 3-OH; verbascoside (5) has four phenolic hydroxyl groups between both hydroxytyrosyl and caffeoyl units.

Evaluation of Antioxidative Activity of Isolated Phenolics
Phenolic compounds are believed to be scavengers of free radicals. Their antioxidative activity depends on their chemical structure; specifically, it depends on their ability to donate hydrogen or electron from the aromatic structure. Different antioxidants respond differently in different measurement methods.

Hydrogen Peroxide Scavenging Activity
Biological systems can generate H 2 O 2 , which is known to be a non-radical oxidant that is not very reactive. It is sometimes toxic and induces cell death because it can form the hydroxyl radical HO·, which is the most potent and reactive species [27]. Accordingly, the effective scavenging of H 2 O 2 can prevent the oxidative damage of cells.
In our preliminary screening, tyrosyl acetate (1) and (8E)-ligustroside (3) did not exhibit any significant H 2 O 2 scavenging activity. Owing to the structural difference, the activity was adopted as molar concentration. The IC 50 of the test compounds (expressed in μM) revealed that the activity followed the order gallic acid > (+)-phillygenin (2) > verbascoside (5) > quercetin > Trolox > rutin (4) ( Table 2). These results demonstrate that the compounds that contain having catechol or a methylated-catechol group-which were 2, 4, and 5-exhibited greater H 2 O 2 activity than compounds that contained one phenolic hydroxyl group-which were 1 and 3. This finding is consistent with the study of Ozyurek et al. [28]. Among the three isolated phenolics, 2, 4, and 5, the strongest H 2 O 2 scavenger was (+)-phillygenin (2) while the weakest one was rutin (4). Even though all three compounds were less active than the reference gallic acid, compounds 2 and 5 exhibited stronger H 2 O 2 scavenging activity than quercetin and Trolox. Rutin (4) exhibited lower activity than its aglycone, quercetin. The strong activity of (+)-phillygenin (2) suggested that the methoxyl groups on the phenyl ring were more effective than the free hydroxyl group in scavenging H 2 O 2 .

Plant Material
The dried flowers of Gomphrena globosa, Helichrysum bracteatum, Chrysanthemum morifolium, Momordica grosvenori, Chrysanthemum indicum, and Osmanthus fragrans were purchased from a traditional market at Guilin, Guangxi Province, China, in 2007. The dried flower of Nelumbo nucifera and green tea were purchased from a market at Liujia, Tainan County, Taiwan, in 2008. Voucher specimen of Osmanthus fragrans (HCY080801) has been deposited at the herbarium of the Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan.

Extraction of Seven Flowers and Green Tea
One gram of the above seven flowers, G. globosa, H. bracteatum, C. morifolium, M. grosvenori, C. indicum, N. nucifera, and O. fragrans together with green tea (for comparison) was individually extracted with MeOH (30 mL) for 12 h. The filtration was concentrated under reduced pressure to give methanol extract for subsequent antioxidative activity assay.

DPPH Radical Scavenging Assay
This DPPH assay was investigated by the modified method of Shimada et al. [29]. MeOH (3.8 mL), sample solution in methanol (0.2 mL, 1 mg/mL), and 1 mM DPPH solution (1.0 mL) were well mixed and left to stand in the dark at room temperature for 30 min. The final concentration of the sample is 40 μg/mL. The absorbance at 517 nm was measured Sample in methanol was used as blank, while DPPH radical in methanol solution was used as a control. Then, the DPPH radical scavenging activity was calculated according to the following equation: % of DPPH radical scavenging activity = [1 − (A sample − A blank )/A control ] × 100 The concentration providing 50% inhibition (IC 50 ) was calculated from the plot of inhibition percentage against sample concentration by linear regression analysis.

Hydrogen Peroxide Scavenging Assay
Hydrogen peroxide (H 2 O 2 ) scavenging activity was measured by the modified method of Sroka and Cisowski [30]. Sample solution in water (100 μL, 1 mg/mL), 0.002% H 2 O 2 (100 μL) and 0.1 M phosphate buffer (Na 2 HPO 4 :KH 2 PO 4 , pH 7.4, 0.8 mL) containing 100 mM NaCl were mixed thoroughly. Then 0.3 mg/mL phenol red (1 mL) with horseradish peroxidase (0.2 mg/mL) 0.1 M phosphate buffer was added. After 15 min at room temperature, a solution of 1 M NaOH (50 μL) was added and the absorption at 610 nm was measured immediately. Water solution without sample was used as a control. Then, the H 2 O 2 scavenging activity was calculated according to the following equation: % of H 2 O 2 scavenging activity = [(A control -A sample )/A control ]  100 The concentration providing 50% inhibition (IC 50 ) was calculated from the plot of inhibition percentage against sample concentration by linear regression analysis.

Determination of Total Phenolic Content
According to the method described by Yen and Hung [31], sample solution in methanol (0.1 mL, 1 mg/mL) was well mixed with 2% Na 2 CO 3 (2 mL). After an interval of 3 min, 50% Folin-Ciocalteau reagent (0.1 mL) was added. The mixture was allowed to stand at room temperature 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 equivatlents (mg of GAE per g of sample).

Statistical Analysis
Each value was expressed as a mean ± SD. At least triplicate experiments were conducted. We analyzed the variance using ANOVA followed by Duncan's multiple range test to determine which means were significantly different from each other or the control. In all cases, a p value of <0.05 was used to determine the significance.