HPLC Analysis and Antioxidant Evaluation of Acteoside-Rich Osmanthus fragrans Extracts

College of Pharmacy, Mokpo National University, 1666 Yeongsan-ro, Muan-gun, Jeollanam-do 58554, Republic of Korea Department of Biomedicine, Health & Life Convergence Sciences, BK21 Four, College of Pharmacy, Mokpo National University, Jeonnam 58554, Republic of Korea College of Pharmacy, Pusan National University, 2 Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 46241, Republic of Korea Jeollanam-do Forest Resource Research Institute, 7 Dado-ro, Sanpo-myeon, Naju-si, Jeollanam-do 58213, Republic of Korea Research and Development Institute, CY Pharmaceutical Co., 340 Nonhyeon-ro, Gangnam-gu, Seoul 06227, Republic of Korea School of Dentistry, Chosun University, Gwangju, 460 Iksan-daero, Iksan-si, Jeollabuk-do 54538, Republic of Korea


Introduction
Osmanthus fragrans (Oleaceae) is a well-known ornamental plant in Asia [1]. It is widely cultivated as an ornamental plant for its fragrant flowers in Korea, Taiwan, Japan, China, and elsewhere. O. fragrans has been used for treating weakened vision, asthma, coughs, toothaches, stomachaches, diarrhea, and hepatitis. However, modern evidence for the biomedical use of O. fragrans is limited [2]. Recently, various biological activities of O. fragrans, such as antilipotoxic [3], antihypoxic [4], anti-inflammatory [5], antiallergic [2], and neuroprotective [6] activities, were reported. Zhou et al. reported that the extract of O. fragrans flowers showed antihypoxic activity, and 80% acetone and 95% ethanol (EtOH) extracts contained about 3-7% acteoside ( Figure 1) [4]. Hung et al. reported that the 75% EtOH extract of O. fragrans flowers had an antiallergic effect in a mouse model at an oral dose of 1 g/kg/14 day [2]. According to their results, the extract of O. fragrans flowers could be administered at a dose of about 4.8 g/day to a 60 kg human. However, pharmacologically active substances supporting their results have not been reported; only total phenolic compounds and total flavonoid concentrations have been reported (367 mg/g phenolics and 45 mg/g flavonoids) [7].
Few  [1]. ey used 80% EtOH for the extraction and partially purified the extract with adsorptive macroporous resin to prepare an extract containing 52% acteoside. Extracts of O. fragrans flowers showed no toxicity for 90 days at a dose of 2 g/kg/day. is is equivalent to 90 days of administration of 1 g/kg as acteoside. Based on these results, the acteoside dose calculated for humans is 4.86 g/60 kg [1]. e pharmacological activity of O. fragrans has been reported in several studies. However, there is a lack of research on the origin of the source materials and their association with biologically active compounds. Recently, researchers have reported the biological activities and identified several components of the flower extracts of O. fragrans. Hung et al. identified five compounds, including tyrosyl acetate, (+)-phillygenin, (8E)-ligustroside, rutin, and acteoside from the dichloromethane extract of O. fragrans flowers [7]. Among them, phillygenin, rutin, and acteoside showed strong antioxidant activity. Liu et al. identified 16 compounds from O. fragrans var aurantiacus flowers; among them, ligustroside and (+)-pinoresinol were identified as showing the best inhibition of nitric oxide production in lipopolysaccharideinduced RAW 264.7 cells [8].
We reviewed the results of previous studies extensively and confirmed that acteoside is a major component of O. fragrans and that O. fragrans could be developed as a safe medicinal source. However, the flower parts of O. fragrans are hard to industrialize owing to low yields. In a preliminary study, we found that the main component of O. fragrans was acteoside and that it was present in large amounts in the flowers and leaves. In the present study, we established a quality control method by high-performance liquid chromatography (HPLC), which could separate and quantify acteoside in the extracts of O. fragrans leaves and flowers. To obtain an acteoside-rich extract, leaf and flower extracts were prepared from O. fragrans and O. fragrans var. aurantiacus, which are distinctive plants grown in Korea. Subsequently, the extraction yields, acteoside contents, and antioxidant activities of the leaf and flower extracts were examined. Antioxidant activity was confirmed by measuring the 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, and total phenolic content.

Identification of Acteoside in O. fragrans Extracts.
All analyses were performed on a Waters Alliance 2695 HPLC system with a photodiode array detector. e analytical column used was an Agilent Zorbax extended C18 (5 μm, 150 mm × 5 mm), with the mobile phase consisting of a mixture of solvent A (acetonitrile) and B (water containing 0.2% phosphoric acid) and employing a gradient elution (from 10/90 to 100/0, v/v) at a flow rate of 1 mL/min (Table 1). e column temperature was maintained at 25°C, and the detec- e acteoside standard was purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China) and had a purity of >98% ( Figure 1).

Method Validation.
For the validation of the analytical method used for the quantification of acteoside in the extract of O. fragrans, specificity, linearity, sensitivity, accuracy, precision, and recovery were determined as previously described [9].

DPPH Free Radical
Assay. Antioxidant activity was determined using a DPPH radical-scavenging assay. In brief, 1 mL of sample solution was mixed with DPPH sample solution (0.4 mM). e mixture contents were allowed to react at room temperature for 10 min. e absorbance of the mixture at 517 nm was then measured using a microplate reader (Perkin Elmer, Waltham, MA, USA) [10].

Ferric-Reducing Antioxidant Power (FRAP) Test.
e reducing power of the sample was determined using a modified FRAP test [10]. In brief, the sample was mixed with sodium phosphate buffer (0.2 M) and potassium ferricyanide (1%, w/v) followed by incubation at 50°C for 20 min. Subsequently, trichloroacetic acid solution (10%, w/v) was added to the reaction mixture followed by centrifugation at 12000 ×g for 10 min. e supernatant was mixed with distilled water and iron (III) chloride solution (0.1%). e absorbance of the resulting solution was measured at 700 nm. e reducing power of the samples is expressed as ascorbic acid equivalents.

Determination of Total Phenolic Content.
Total phenolic content was determined using the Folin-Ciocalteu assay.
e sample was mixed with sodium carbonate solution (2%, w/v) and Folin-Ciocalteu's phenol reagent (10%, v/v) for 10 min, and its absorbance was measured at 750 nm. e data are expressed as milligrams of gallic acid equivalents per gram of sample [10].

Statistical Analysis.
e t-test was used for comparing two unpaired means, and a p value <0.05 was considered statistically significant. Data are expressed as mean ± standard deviation.  Table 2, the hot water and 20-100% EtOH extraction yields ranged from 6.12% to 42.22%. e best overall yield was obtained by the hot water extraction of O. fragrans flowers. is study is the first report of the extraction yields determined according to the extraction conditions for two species of O. fragrans. Our data could provide optimal extraction information when O. fragrans is developed as a pharmaceutical material in the future.

Limit of Detection (LOD), Limit of Quantification (LOQ), and Linearity.
e LOQ of an individual analytical procedure is the lowest amount of analyte in a sample that can be determined with suitable precision and accuracy. e LOD and LOQ for acteoside were found to be 0.26 and 0.78 μg/mL, respectively. e LOD and LOQ values derived from this experiment were less than 1 μg/mL and were sufficient for the analysis of O. fragrans. e calibration curve was linear over an acteoside concentration range of 6.25-100 μg/mL. e calibration curves exhibited good linear regression (r 2 � 0.999) ( Table 3).

Precision, Accuracy, and Repeatability.
e intraday and interday precision and accuracy data are shown in Table 4. e developed method was found to be precise, with the obtained RSD values being below 2.0%, and the accuracy was in the range of 98.33-103.42%. e recovery data are shown in Table 5. ese results indicate that the developed method was reproducible with good precision and accuracy.

Acteoside Contents in O. fragrans Extracts.
e acteoside contents in the O. fragrans extracts were determined by HPLC analysis. As shown in Figure 2, acteoside was identified as the main compound in the extract of O. fragrans. We will identify other bioactive substances in this plant in the future. In the present study, we used acteoside as the main target, and tried to develop an acteoside-rich extract as a drug.
e acteoside contents resulting from various extraction conditions are shown in Figure 3. e acteoside content of the O. fragrans extracts tended to be significantly higher in ethanolic extracts than in hot water extracts. All three types of extracts, except OFL, showed more than 10% acteoside content in the 100% EtOH extract. In particular, the content of the 100% ethanol extract of OFVF tended to be the highest (13.86%). OFF also showed a high content, of 11.91%, indicating that the maximum acteoside content can be obtained from flowers. e acteoside contents of the 100% and 80% EtOH extract of OFVL were 10.81% and 11.14%, respectively. In particular, if the leaves are superior to flowers in terms of acteoside content, further research on leaf extracts should be conducted. We found no existing reports on biologically active phytochemicals of O. fragrans leaves.

Antioxidant Properties of O. fragrans Extracts.
e antioxidant activities of various extracts of O. fragrans were determined by measuring the DPPH-scavenging activity, reducing power, and total phenolic content. As shown in Figure 4, the extraction conditions produce higher DPPHscavenging activity in the 40/60/80% EtOH for OFL and 60% EtOH for OFF than in the hot water. e reducing power was tested for 100 μg/mL of each extract and was calculated as ascorbic acid equivalents (μg/mL eq. ascorbic acid). As shown in Figure 5, the extraction condition produce higher reducing power in Journal of Food Quality 3 the 40/60/80/100% EtOH for OFL, 60/80/100% EtOH for OFF, 60/80% EtOH for OFVL, and 60/80% EtOH for OFVF than in the hot water. In particular, the 60 and 80% EtOH extracts of OFVL showed the best reducing power among all extracts (176.83 ± 4.53 μg/mL eq. ascorbic acid). e total phenolic compounds, determined using the Folin-Ciocalteu method, were expressed as gallic acid equivalents, with reference to a standard curve (r 2 � 0.999). e total phenolic contents of the extracts are shown in Figure 6. e extraction conditions produce higher total phenolic content in the 40/60/80/100% EtOH for OFL, 60/80/100% EtOH for OFF, 60/80/100% EtOH for OFVL, and 60/80/100% EtOH for OFVF. Particularly, the 80% EtOH extract of OFVL and the 100% EtOH extract of OFL showed the highest total phenolic content. However, in terms of productivity, the extraction yield of the 100% EtOH extract of OFL (9.53%) was lower than that of the 80% EtOH extract of OFVL (12.41%). us, the 80% extract of OFVL showed the best phenolic content.
In summary, the flower and leaf extracts of O. fragrans and O. fragrans var. aurantiacus were prepared, and their extraction yields, marker compound (acteoside) content, and antioxidant activities were investigated. Overall, almost all extracts    prepared using EtOH-based extraction solvents tended to produce better antioxidant activity than those prepared using hot water. ese results suggest that the ethanolic extract of O. fragrans could serve as a potential antioxidant and antiinflammatory pharmaceutical source, and our validated method would be useful for the quality control of O. fragrans extracts.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.