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Identification and Quantification of Active Components in Osmanthus fragrans Fruits by HPLC-ESI-MS/MS Xiaoyan Liao, Fangli Hu, and Zilin Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05560 • Publication Date (Web): 09 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Journal of Agricultural and Food Chemistry

Graphic abstract 226x134mm (192 x 192 DPI)

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Identification and Quantitation of the Bioactive Components in Osmanthus fragrans Fruits

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by HPLC-ESI-MS/MS

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Xiaoyan Liao,†,‡ Fangli Hu,† Zilin Chen*,†,‡

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†

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Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China

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‡

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China

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and

State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing, 10080,

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*Corresponding author (Tel: +86-27-68759893; Fax: +86-27-68759850; E-mail:

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[email protected])

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ABSTRACT:

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Information on the chemical composition of Osmanthus fragrans fruits is still limited since there

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are many compounds present in low concentrations in the plant. In this work, the bioactive

17

components in O. fragrans fruit extract were investigated by a new HPLC-ESI-MS/MS method,

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which allows sensitive analysis both in identification and quantitation. A total of twenty-eight

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compounds were tentatively identified, and sixteen components were discovered in O. fragrans

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fruits for the first time. The validated quantitative methods for the determination of the bioactive

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components were subsequently applied to analyze batches of O. fragrans fruits from different

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cultivars, which will be beneficial for the comprehensive utilization of O. fragrans fruits.

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KEYWORDS: Osmanthus fragrans fruits, HPLC-ESI-MS/MS, identification, quantitation,

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bioactive components, phenylethanoid glycosides



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INTRODUCTION

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Osmanthus fragrans Lour. (O. fragrans), an ornamental plant, is well known for its fragrance and

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is widely cultivated in Asia, especially in southern and central China. The essential oil of the

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flowers is used in aromatherapy and to produce perfumes,1 and the flowers have been consumed

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for a long time as additives in food, tea, and other beverages to improve their taste. In recent

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research, since O. fragrans flowers contain large amounts of bioactive components, such as

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phenolic acids, it has great potential to be an ingredient in functional food due to its natural

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antioxidants.2-4

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O. fragrans is androdioecious,5 and the hermaphrodite cultivars can bear fruits. The

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easily plucked flowers are suitable for use in perfume production and as an additive for food.

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However, ripe O. fragrans fruits, which are composed of a purple peel, green pulp and light

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brown seed, are always considered waste. Previous studies have shown that the extracts and

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fractions of O. fragrans fruits have anti-inflammatory,6 antioxidative activities7-10 and inhibitory

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effects on platelet aggregation,11 which means that the large amount of O. fragrans fruit ‘waste’

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has potential applications, such as functional food ingredient for health products. Therefore,

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accurate identification and quantitation of the bioactive components in O. fragrans fruits is

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necessary and important if the whole fruit is to be used efficiently.

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In recent years, several studies on the phytochemicals of O. fragrans fruits have been

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reported. A few bioactive compounds12-17 including natural melanin, red pigment, oleoside

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dimethyl ester, salidroside, nuezhenide and Gl3, were isolated from peels, pulps and seeds.

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Twenty-three compounds from the ethyl acetate extract of O. fragrans fruits18 and ten secoiridoid 3 ACS Paragon Plus Environment

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glucosides from the n-butanol extract11 were found respectively, but information on the chemical

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composition of O. fragrans fruits is still limited. The above isolation and purification methods

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were mainly focused on column chromatography, which is laborious, especially for the

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separation of those present in low concentrations and those that co-elute. HPLC-MS/MS

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combines advantages like the high separation efficiency of LC and the high sensitivity and

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molecular structure elucidation of MS, and it has become a powerful technique for the

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identification of bioactive natural products because of its operational simplicity, low cost and

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high speed.19

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In this work, the bioactive components in O. fragrans fruit extracts were investigated by

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HPLC-ESI-MS/MS. The MS/MS system was based on a triple quadrupole, and the third

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quadrupole was equipped with a linear ion trap. This method is sensitive for both the

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identification (Enhance Product Iron scan, EPI) and quantitation (Multiple Reaction Monitoring

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scan, MRM) of the components. A total of twenty-eight compounds were tentatively identified

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and sixteen components were discovered in O. fragrans fruits for the first time. The validated

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quantitative methods for the determination of the bioactive components were subsequently

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applied to analyze batches of O. fragrans fruits from different cultivars, which will be beneficial

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for the efficient utilization of O. fragrans fruits.

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MATERIALS AND METHODS

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Chemicals and reagents 4 ACS Paragon Plus Environment


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Salidroside, nuezhenide, neonuezhenide, acteoside, oleanolic acid and ursolic acid were

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purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Isoacteoside was

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purchased from Chengdu Ruifensi Biotechnology Co., Ltd. (Sichuan, China). HPLC-grade

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acetonitrile and methanol were purchased from Fisher Scientific (Fairlawn, NJ). Formic acid and

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ammonia were purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Ultrapure

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water was purchased from Hangzhou Wahaha Group Co., Ltd. (Zhejiang, China). All other

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reagents and chemicals were of analytical grade.

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O. fragrans fruits were collected from Wuhan, China in May 2016. Ten batches of O.

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fragrans fruits (Whu-X-1, Whu-X-2, Whu-X-3, Whu-X-4, Whu-X-5, Whu-XS-1, Whu-Y-1,

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Whu-Y-2, DH-1 and ZS-1) are labeled No.1-10 successively. They are preserved in herbaria of

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Wuhan University School of Pharmaceutical Sciences, and identified by prof. Jun Tang of

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Wuhan University School of Pharmaceutical Sciences. Batch 1, 7, 8 and 10 of them belong to O.

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fragrans Albus group and the others belong to O. fragrans Luteus group. Samples were dried at

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60 °C in an oven, pulverized into powders, and then stored at -20 °C until further use.

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Preparation of samples and standard solutions

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The following method was used to prepare samples for identification: 1 g of the dry O. fragrans

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fruit powder was extracted with 100 mL of 70% methanol in an ultrasonic water bath for 45 min

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at room temperature. The methanol was evaporated from the extract under reduced pressure at

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60 °C. Then, the residue was suspended in 5 mL of water and extracted three times each with 5

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mL of petroleum ether, ethyl acetate and n-butanol sequentially. The n-butanol was evaporated 5 ACS Paragon Plus Environment

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from the corresponding extract under reduced pressure. The resulting residue was dissolved in 5

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mL of methanol.

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The following method was used to prepare samples for quantitative analysis: for

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phenylethanoid glycosides, 0.1 g of the O. fragrans fruit powder was extracted with 10 mL of 70%

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methanol in an ultrasonic water bath for 45 min at room temperature. A 100-µL aliquot of the

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sample was diluted to 1 mL with a methanol/water solution (10:90, v/v). For triterpenes, the

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extraction solvent was optimized to 12 mL of 100% methanol instead. A 100-µL aliquot of the

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sample was diluted to 1 mL with 0.01% aqueous ammonium hydroxide/methanol (22:78, v/v).

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All extracted samples were filtered through a 0.22-µm membrane filter before injection.

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For HPLC quantitation, a stock solution of seven reference substances was prepared in concentrations ranging from 454 to 525 µg/mL in methanol and stored at 4 °C until use. Standard

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working solutions of the mixtures were obtained by diluting stock solutions to the desired

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concentrations.

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HPLC-MS/MS analysis

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HPLC-MS/MS analysis was performed on a LC-20AD series chromatographic system

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(Shimadzu, Kyoto, Japan) coupled with an API4000Qtrap mass spectrometer equipped with an

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electrospray interface (ESI) (AB Sciex, Framingham, MA). Equipment control and data analysis

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were performed using Analyst software ver. 1.6.2 (AB Sciex). The HPLC instrument included

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two LC-20AD binary pumps, a DGU-20A degasser, an SIL-20AC autosampler at 15 °C and a

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CTO-20AC column oven at 40 °C. The injection volume for all samples was set at 1 µL. 6 ACS Paragon Plus Environment


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The main parameters in mass spectrometry were the ion spray voltage, -4500 V (4500 V

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in positive ion mode); temperature, 550 °C; curtain gas, 35 psi; nebulizer gas, 50 psi; heater gas,

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50 psi; and collision gas, Medium (High in EPI scan).

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High resolution MS data were obtained by an UltiMate 3000 Rapid Separation Liquid

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Chromatography system (Thermo Scientific, Germering, Germany) coupled with a Q Exactive

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Focus mass spectrometer (Thermo Scientific, Waltham, MA) controlled by the Xcalibur 3.0

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software (Thermo Scientific).

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Identification of bioactive components

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Chromatographic separations were carried out on an Ultimate C18 column (50 mm×2.1 mm, I.D.,

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5 µm, Welch Technologies, Shanghai, China). The optimum HPLC separation gradient was

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determined for the most efficient separation. The flow rate was 0.5 mL/min, and the solvent

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system was water with 0.1% aqueous formic acid (A) and acetonitrile (B). The gradient was

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operated as follows: 0-3 min, 5-10% B; 3-10 min, held at 10%B; 10-20 min, 10-16% B; 20-40

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min, 16-50% B; 40-50 min, 50-90% B; 50-55 min, held at 90%B; 55-60 min, held at 5%B.

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For MS detection, mass spectra in negative ion mode and positive ion mode were

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recorded in the m/z 200 –1200 range with accurate mass measurements of all mass peaks. The

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MS/MS spectra were acquired by EPI scans in negative ion mode at CE of -40 V with CE spread

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of -15 V. The declustering potential for both scan types was set at -80 V.

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Determination of phenylethanoid glycosides and triterpenes Determination of phenylethanoid glycosides. All separations were performed on an 7 ACS Paragon Plus Environment

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Ultimate C18 column (50 mm×2.1 mm, I.D., 5 µm, Welch Technologies, Shanghai, China) at

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40 °C with a flow rate of 0.4 mL/min. A mobile phase consisting of methanol/water (10:90, v/v)

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(A) and 0.05% methanolic formic acid (B) was used in a multistep gradient mode. The optimized

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gradient was as follows: 0-4 min, held at 0% B; 4-15 min, held at 20%B; 15-18 min, 20-90% B;

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18-23 min, held at 90%B; 23-35 min, held at 0%B.

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Determination of triterpenes. A C18-H column (250 mm×4.6 mm, I.D., 5 µm, Amethyst,

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U.S.) was used for isocratic elutions. The flow rate was 0.5 mL/min. The mobile phase was 0.01%

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aqueous ammonium hydroxide/methanol (22:78, v/v).

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RESULTS AND DISCUSSION

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Characterization of the bioactive components in O. fragrans fruit extract

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The bioactive components in O. fragrans fruit extract were investigated by HPLC-ESI-MS/MS.

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Both negative and positive ion modes were employed to identify the corresponding signals. As

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shown in Figures 1 and 2 and Table 1, twenty-eight compounds were tentatively assigned from

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the n-butanol extracts of the O. fragrans fruits. Sixteen components (1, 3, 5, 8, 10, 11, 13, 14, 16,

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17, 19, 20, 21, 23, 25, and 26) were discovered in O. fragrans fruits for the first time. Owing to

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the sensitive analysis by EPI scan, four new isomers of previously described compounds were

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found, including glucopyranosyl methyloleoside isomer, 5, nuezhenide isomer 3, 20, and two

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Gl3 isomers, 23 and 25. Other twelve compounds including hydroxytyrosol glucoside, 1,



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caffeoyl rhamnosylglucoside, 3, β-Hydroxyacteoside, 8, excelside A, 10, neonuezhenide, 11,

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acteoside, 13, isoacteoside, 14, methoxy nuezhenide, 17, two glucopyranosyl dimethyloleoside

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isomers, 16 and 19, methyloleoside-neonuezhenide, 21, and Gl5 isomer, 26, have not been

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described in either the pulps or seeds, and were discovered in O. fragrans fruits for the first time.

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Moreover, compound 21 (methyloleoside-neonuezhenide) is a new compound and was reported

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for the first time. The assignments were corroborated by the results of high-resolution MS to

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confirm the accurate molecular weight of these compounds. Triterpenes and phenolic compounds

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such as secoiridoid and phenylethanoid glucosides were shown to be the main components.

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It was found that the phenylethanoid glucosides and secoiridoid derivatives appear

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primarily as the deprotonated molecular ions ([M-H]-) and adduct molecular ions ([M+ formic

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acid-H]-) in negative ion mode, and they appear as the adduct molecular ions ([M+NH4]+ and

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[M+Na]+) in positive ion mode. Most of the compounds were derivatives of three structural units,

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hydroxyphenethyl alcohol, glucose, and

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3-ethylidene-3,4-dihydro-2-hydroxy-5-(methoxycarbonyl)-2H-pyran-4-acetic acid (aglycone of

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methyloleoside), combined with different linkages.

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Phenylethanoid glucosides derivatives

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Salidroside, 2, which appears at a retention time (tR) of 1.94 min, has been already described as

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the most effective antioxidant in O. fragrans pulp.14 It exhibited a parent ion at m/z 299.2, which

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generated major fragments at m/z 119.0 and m/z 137.0 by loss of a glucose (180 Da) or glucose

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residue (162 Da). The identity of 2 was confirmed by comparison to an authentic standard. The 9 ACS Paragon Plus Environment

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deprotonated molecular ion of 1 (at m/z 315.2), identified as hydroxytyrosol glucoside, showed a

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loss of 162 Da or 180 Da to generate major ions at m/z 153.2 and m/z 135.2, suggesting the

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presence of an hydroxytyrosol moiety.20 Acteoside, 13, and isoacteoside, 14, showed

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characteristic fragments (m/z 179.1, m/z 161.1 and m/z 135.0) of a caffeoyl group, and they were

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confirmed by comparing retention times with that of commercial standards. β-Hydroxy acteoside,

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8, was observed at m/z 639.6 and showed the typical fragments of a caffeoyl group. The

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fragment at m/z 621.0 could be attributed to the loss of H2O.21 Compound 3 was observed at m/z

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487.2, suggesting a formula that was less than that of acteoside by a C8H8O2 group which is

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equivalent to the dehydrated residue of hydroxytyrosol (136 Da). The typical fragments of a

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caffeoyl group were observed in its MS2 spectra, so compound 3 was tentatively assigned as

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caffeoyl rhamnosylglucoside.

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Methyloleoside derivatives

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Compound 6 (oleoside-11-methyl ester) and compound 7 (secoxyloganin) were observed at m/z

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403.1(tR 4.34 min and 6.63 min). They have been separated and described in the literature.11

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They both showed ions at m/z 371.1 and m/z 223.1, which were attributed to neutral losses of

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CH3OH and glucose, respectively. The typical fragment ions of methyloleoside aglycone (m/z

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223.1, m/z 191.1, m/z 181.1, and m/z 149.1)22,23 were observed in the MS2 spectra of 6 and 7.

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Compound 9 exhibited a deprotonated molecular ion at m/z 417.4. The major fragment at m/z

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185.1 was due to the neutral loss of C4H6O from the fragment ion at m/z 255.2, further producing

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a fragment at m/z 153.1 by loss of CH3OH, which is characteristic of an oleoside dimethyl 10 ACS Paragon Plus Environment


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ester.24 Compound 10 (tR 10.01 min, m/z 579.7), assigned as excelside A,25 was found to have a

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formula that was 162 Da greater than oleoside dimethyl ester. The typical fragments of oleoside

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dimethyl ester were obtained at m/z 255.1, m/z 223.1, m/z 185.1 and m/z 153.1. The fragment ion

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at m/z 323.1 suggested the glucose moiety was located on another glucose moiety to form a

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diglucoside residue. The ions observed at m/z 565.2 (tR 3.48, 3.72 min) were assigned as

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glucopyranosyl methyloleoside isomers 4 and 5. The major fragment ion at m/z 403.2 was

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obtained through the loss of a glucose moiety, which subsequently gave rise to the ions at m/z

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371.2, m/z 223.1 and m/z 179.2. Those ions were indicative of a methyloleoside subunit.

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Similarly, compound 16, assigned as glucopyranosyl di-methyloleoside, exhibited a deprotonated

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molecular ion at m/z 951.7, which was 386 Da greater than the ion at m/z 565, suggesting the

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further formation of a methyloleoside derivative. The typical fragment ions of methyloleoside

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(m/z 371.2, m/z 223.1 and m/z 179.2) were generated in the MS2 spectra. Compound 19, also

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observed at m/z 951.7, is an isomer of compound 16.

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Derivatives based on both phenylethanoid and methyloleoside

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Nuezhenide (15, tR 18.20 min) and Gl3 (24, tR 26.24 min) were found to be the major

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components of O. fragrans seeds16 and had at m/z 685.6 and m/z 1071.6, which were the most

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intense chromatographic peaks. Three nuezhenide isomers, 12, 18 and 20), were detected at tR

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20.28, 22.92, and 24.30 min, respectively. Nuezhenide and its isomers exhibited similar

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fragmentation patterns. The fragment at m/z 523.2 was attributed to the loss of a glucose moiety,

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which afforded the major fragments at m/z 453.2 and m/z 421.2 by further neutral loss of C4H6O 11 ACS Paragon Plus Environment

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(70 Da) and double neutral loss of C4H6O and CH3OH (32 Da). The ions at m/z 299.1, m/z 179.1,

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and m/z 119.1 were the typical fragments of a salidroside unit. In addition, the ions at m/z 223.1,

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m/z 191.1, and m/z 149.1 were the typical fragments of methyloleoside aglycone. The major

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fragments of Gl3 were at m/z 839.3, m/z 685.3, m/z 523.1, m/z 453.1, m/z 421.3 and m/z 299.2.

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Two Gl3 isomers (23 and 25) were detected at 26.19 min and 27.77 min. Gl3 isomers are the

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methyloleoside derivatives of nuezhenide, and their MS2 spectra were compared to those

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reported in the literature.20 Other nuezhenide derivatives were found with m/z 701.7 (11) and m/z

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715.7 (12); these compounds shared the same fragmentation pattern and were assigned as

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neonuezhenide26 and methoxy nuezhenide,27 respectively. The fragments of the ion at m/z 701.7

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(m/z 315.1, m/z 297.2 and m/z 135.1) were 16 Da greater than the typical fragments (m/z 299.1,

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m/z 281.2 and m/z 119.1), suggesting the hydroxy group was located on the phenylethanol

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moiety; the product ion that arose from the deprotonated molecular ions at m/z 715.7 were 30 Da

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greater than the typical fragments, suggesting the presence of a methoxy group. The identities of

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nuezhenide and neonuezhenide were also confirmed by comparison to authentic standards.

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Compound 21 exhibited a deprotonated molecular ion at m/z 1087.4 with fragment ions typical

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of neonuezehenide (m/z 701.3, m/z 469.2, m/z 437.2 and m/z 315.2). The major fragment ion at

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m/z 701.3 was attributed to the loss of a methyloleoside moiety, so 21 was assigned as

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methyloleoside-neonuezhenide. The fragments at m/z 925.3 and m/z 855.3 were generated by the

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loss of a glucosyl moiety and followed by neutral loss of C4H6O (70 Da). Moreover,

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Methyloleoside-neonuezhenide, 21, is a new compound and was reported for the first time.

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Another derivative was observed at m/z 523.5 (22, tR 24.80 min), and was assigned as 12 ACS Paragon Plus Environment


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ligstroside. The major fragment at m/z 291.1 was obtained by the loss of a glucose residue and

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subsequent loss of C4H6O.28 The MS2 spectra of compound 26 (m/z 909.6) suggested it was also

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based on a ligstroside structure because the characteristic fragments (m/z 291.1, m/z 259.0 and

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m/z 223.0) were observed. The ion at m/z 677.2 is likely due to successive loss of a glycose

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moiety and C4H6O. There was no fragment at m/z 839, which indicated that the glycosides are on

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the termini of the structure. According to this limited amount of information, 26 can only can be

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proposed to be an isomer of Gl5.29,30

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Triterpenes

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Oleanolic acid, 27, and ursolic acid, 28, were confirmed based on comparison to commercial

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standards, and for both compounds, there was only one major product ion observed at m/z 407.2.

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Overall, the fragments of phenylethanoid glucosides could be produced by neutral losses

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of glucose (180 Da) and glucose residues (162 Da), and product ions at m/z 119

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(4-hydroxyphenethyl alcohol) or at m/z 135 (3,4-dihydroxyphenylethanol) could be observed in

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the MS/MS spectra. If there was a subunit of methyloleoside, the fragments could be produced

250

by the neutral loss of 224 Da or 386 Da, and a typical product ion at m/z 223 would be observed.

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According to the above results, compared with previously reported methods,11-18

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HPLC-ESI-MS/MS is a simple and efficient method to discover a series of bioactive components

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contained similar structural units. The isolation and purification of the individual components

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can be avoided especially for those present in low concentrations and those that co-elute.

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Optimization of extraction and analytical conditions

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To extract the phenylethanoid glycosides and triterpenes efficiently, the variables involved in the

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procedure such as the extraction solvent (60%, 70%, 80%, 90% and 100% methanol), the volume

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of extraction solvent (6 mL, 8 mL, 10 mL, 12 mL, 14 mL), and extraction time (15, 30, 45 and

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60 min) were optimized. The final optimized method for phenylethanoid glycosides was as

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follows: 0.10 g of the O. fragrans fruit powder was extracted with 10 mL of 70% methanol in an

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ultrasonic water bath for 45 min at room temperature. For triterpenes, the extraction solvent was

263

optimized to 12 mL 100% methanol instead.

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For the MS conditions, the collision energy, declustering potential, and collision cell exit

265

potential were optimized for the quantitative analysis of the contents of O. fragrans fruits. The

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product ions with high responses were chosen. The HPLC conditions, including type of column,

267

injection volume, and mobile phase system, were optimized. Three different brands of columns,

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namely, Amethyst C18-H, Amethyst C18-P and Welch Ultimate C18, were used. The Ultimate

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C18 column most efficiently separated the five phenylethanoid glycosides, and the Amethyst

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C18-H column was more suitable for the separation of the two triterpenes. In addition, different

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mobile phases, such as acetonitrile and methanol with a variety of modifiers (including formic

272

acid, ammonium acetate, and ammonia), were tested. It was found that the sensitivity for the

273

salidroside was low when formic acid was added to the mobile phase, while it was high when the

274

mobile phase was methanol and water. Acteoside and isoacteoside had high responses only when

275

formic acid was present in the mobile phase, and neonuezhenide and nuezhenide showed good

276

responses both with and without formic acid. Mobile phase A was optimized to be a mixture of 14 ACS Paragon Plus Environment


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water and methanol (90:10, v/v). When salidroside was eluted, mobile phase B (0.05% formic

278

acid in methanol) was mixed with mobile phase A to improve the sensitivity for acteoside and

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isoacteoside. The retention times for oleanolic acid and its isomer, ursolic acid, were too long in

280

acidic or neutral mobile phases for baseline resolution with good response. High sensitivity with

281

short retention times were found when a certain amount of ammonia was added to the mobile

282

phase. Therefore, phenylethanoid glycosides and triterpenes were detected separately.

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Method validation

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Linear calibration curves were obtained by plotting peak areas versus concentrations at different

286

levels. All the calibration curves showed good linearity within the experimental range with

287

correlation coefficients (r) larger than 0.9997. The limits of quantitation (LOQs) under the

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present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 10. The

289

results shown in Table 2 suggest that the HPLC-ESI-MS/MS method for the simultaneous

290

detection of five phenylethanoid glycosides (salidroside, acteoside, isoacteoside, neonuezhenide

291

and nuezhenide) and the determination of oleanolic acid and ursolic acid has excellent sensitivity

292

compared to other literature methods.31-34

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Intra-day and inter-day precision were investigated by injecting freshly prepared mixtures

294

of standards five times on the same day and on 3 consecutive days, respectively. In order to test

295

the repeatability of the method, nine samples at three different concentrations of 70%, 100%, and

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130% levels (three samples at each level) were prepared and analyzed by extracting from 0.07 g,

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0.1 g and 0.13 g of the O. fragrans fruit powder from the same batch. For stability test, the 15 ACS Paragon Plus Environment

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extracted sample were stored at 15 °C and analyzed after 0, 4, 8, 12, 15 h. The results of the

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precision, repeatability and stability evaluations of the method were satisfactory and are shown

300

in Table 2.

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The accuracy of the apparatus was evaluated by adding a known amount of mixed

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reference substances to 0.05 g of the O. fragrans fruit powder which was then extracted as

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described in the sample preparation. As shown in Table 2, the developed analytical method was

304

accurate with recoveries of 90.4–104.5%, with the exception of neonuezhenide, which may be

305

related to its stability in the ultrasonic bath.

306 307

Application in the analysis of real samples

308

The contents of five phenylethanoid glycosides and two triterpenes were measured by the

309

developed HPLC-ESI-MS/MS methods in O. fragrans fruits. The typical chromatograms are

310

shown in Figure 3, and the contents are summarized in Table 3. The contents of the two

311

triterpenes among the ten batches were relatively stable, but there were substantial differences in

312

the contents of the five phenylethanoid glycosides. The contents of noenuezhenide, acteoside and

313

isoacteoside were low in the fruits, and the latter two are the main components of the flowers.35

314

Nuezhenide was one of the main components of the fruits,16 and it is converted to salidroside

315

after being processed as a wine.36 Simultaneous detection of phenylethanoid glycosides such as

316

salidroside and nuezhenide is preferable for the quality control of O. fragrans fruits.

317 318

There are 166 O. fragrans cultivars, and they are split into 4 groups, O. fragrans Asiaticus group, O. fragrans Albus group, O. fragrans Luteus group, and O. fragrans 16 ACS Paragon Plus Environment


319

Aurantiacus group,37,38 based on different flowering seasons, inflorescence types and flower

320

colors. There are a few cultivars in each group that can bear fruits; however, fruit-bearing species

321

are relatively more common in O. fragrans Albus and O. fragrans Luteus group. Interestingly, it

322

was found that the contents of salidroside and nuezhenide in the four batches from O. fragrans

323

Albus group are generally lower than the contents found in the batches collected from O.

324

fragrans Asiaticus group. The differences in the contents may be because the ten batches of O.

325

fragrans fruits belong to different cultivars.

326

Individual compounds including salidroside,39 acteoside,40 isoacteoside,41

327

neonuezhenide,42 nuezhenide,43,44 oleanolic acid45 and ursolic acid46 have been shown to possess

328

a variety of pharmacological activities such as antioxidation, antiaging, anti-obesity,

329

anti-inflammation, anticancer, antiviral, central nervous system inhibition and hepatoprotective

330

activities. Since there are many bioactive components in the O. fragrans fruits, it is valuable to

331

further study the potential functional activities and possible practical applications of this material

332

to utilize overlooked O. fragrans fruits more efficiently.

333 334

ASSOCIATED CONTENT

335

Supporting information

336

This material is available free of charge via the Internet at http://pubs.acs.org.

337

MS2 spectra of compounds 1-28 and MRM scan parameters for the determination of

338

phenylethanoid glycosides and triterpenes.

339 17 ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35


340

AUTHOR INFORMATION

341

Corresponding author

342

* Tel: +86-27-68759893; Fax: +86-27-68759850; E-mail: [email protected]

343 344

Funding

345

This work was supported by the National Natural Science Foundation of China (Grant nos.

346

21375101, 81573384 and 91417301), the Natural Science Foundation of Hubei Province (No.

347

2014CFA077), the Open Project of Wuhan University Facility Division (WHU-2017-SYKF-12)

348

and the Large-scale Instrument and Equipment Sharing Foundation of Wuhan

349

University(LF20170843).

350 351

Note

352

The authors declare no competing financial interest.

353



354

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FIGURE CAPTIONS Figure 1. Total ion chromatogram in negative ion mode of n-butanol extract of O. fragrans fruits Figure 2. Chemical structures of main components identified in the extract of O. fragrans fruits. (Glc: β-D-glucopyranosyl; Caff: Caffeyl; Rha:Rhamnosyl) Figure 3. Typical MRM chromatograms of five phenylethanoid glycosides and two triterpenes in: (A and B) standard solutions; and (C and D) extracts. Peak identification: 2. Salidroside, 11. Neonuezhenide, 13. Acteoside, 14. Isoacteoside, 15. Nuezhenide, 27. Oleanolic acid, 28. Ursolic acid



Page 28 of 35

Tables Table 1. Characterization of the Chemical Constituents of O. fragrans Fruits by HPLC-ESI-MS/MS [M-H]- d No.

tR

[M-H] Tentative assignment

(min)

-c

MS/MS c (m/z)

(m/z)

Exptl

Calcd

Error

(m/z)

(m/z)

(ppm)

1a

1.23

Hydroxytyrosol glucoside

315.2

153.2 (34), 135.2 (32), 123.2 (25), 101.1 (2)

315.1090

315.1075

4.8

2b

1.94

Salidroside

299.2

137.0 (11), 119.0 (100)

299.1139

299.1126

4.3

3a

2.60

487.2

179.2 (100), 161.3 (19), 135.2 (26)

487.1463

487.1446

3.5

565.1780

565.1763

3.0

565.1779

565.1763

2.8

403.1248

403.1235

3.2

403.1248

403.1235

3.2

639.1938

639.1920

2.8

417.1402

417.1392

2.4

579.1935

579.1920

2.6

701.2305

701.2288

2.4

685.2353

685.2338

2.2

623.1986

623.1971

2.4

Caffeoyl rhamnosylglucoside Glucopyranosyl

4

3.48

403.2 (100), 371.2 (7), 265.2 (25), 223.1 (32), 195.2 (26), 565.2

methyloleoside

5a

179.2 (14),

Glucopyranosyl 3.72

403.2 (100), 371.2 (10), 265.2 (16), 223.1 (32), 195.2 (13), 565.2

methyloleoside isomer

179.2 (6) 371.1 (75), 223.1 (100), 191.1 (67), 181.1 (17), 179.1 (58),

6

4.34

Oleoside-11-methyl ester

403.1 149.1 (41), 121.3 (33)

7b

371.1 (100), 333.0 (3), 223.1 (8), 191.1 (8),181.1 (6), 179.1 6.63

Secoxyloganin

403.1 (6), 149.1 (4), 121.1 (10)

8a

621.0 (1), 459.2 (4), 245.1 (1), 233.0 (3), 179.1 (33), 161.1 7.86

β-Hydroxyacteoside

639.6 (100), 151.0 (14),135.1 (7) 385.1 (3), 255.1 (9), 223.1 (16), 185.1 (100), 163.1 (9),

9

8.72

Oleoside dimethyl ester

417.4 153.1 (21)

10 a

547.2 (21), 399.2 (15), 323.1 (12), 255.2 (27), 223.1 (18), 10.01

Excelside A

579.7 185.1 (100), 153.1 (24)

11 a,b

477.1 (2), 469.2 (4), 437.1 (4), 357.1 (2), 315.1 (100), 14.96

Neonuezhenide

701.7 297.2 (7), 191.1 (2), 149.1 (3), 135.1 (8) 461.1 (27), 453.1 (100), 421.1 (32), 299.1 (23), 223.1 (68),

12

16.48

Nuezhenide isomer 1

685.6 179.1 (41), 161.1 (73), 149.1 (50)

13 a,b

461.1 (8), 315.2 (4), 297.3 (1), 179.1 (4), 161.1 (100), 16.59

Acteoside

623.6 135.0 (2)


Page 29 of 35


14 a,b

461.1 (15), 315.2 (8), 297.3 (4), 179.1 (8), 161.1 (100), 18.03

Isoacteoside

623.6

623.1989

623.1971

2.9

685.2352

685.2338

2.0

951.2996

951.2976

2.1

715.2463

715.2444

2.6

685.2353

685.2338

2.2

951.2994

951.2976

1.9

685.2356

685.2338

2.6

1087.3521

1087.3501 1.8

523.1825

523.1810

1071.3572

1071.3551 2.0

1071.3562

1071.3551 1.0

1071.3571

1071.3551 1.9

909.3038

909.3023

1.6

135.0 (5) 523.2 (1), 453.2 (100), 421.1 (36), 393.2 (4), 369.1 (3), 15

b

18.20

Nuezhenide

685.6

299.2 (20), 281.2 (3), 223.1 (20), 191.2 (7), 179.1 (10), 163.1 (7), 149.0 (10), 121.1 (6), 101.0 (9)

16 a

Glucopyranosyl 19.02

789.1 (5), 403.1 (100), 371.1 (45), 315.2 (24), 265.0 (41), 951.2

dimethyloleoside isomer 1

17 a

223.1 (82), 179.1 (45), 161.1 (17), 149.1 (10) 553.2 (3), 483.2 (100), 451.2 (33), 329.2 (30), 311.1 (3),

19.87

Methoxy nuezhenide

715.7 223.2 (18), 205.0 (7), 149.1 (11), 121.0 (6), 101.0 (5) 453.2 (24), 299.2 (21), 265.0 (9), 223.1 (100), 205.1 (54),

20.28

18

Nuezhenide isomer 2

685.6

191.2 (39), 179.3 (36), 153.1 (24), 149.0 (42), 137.2 (36), 121.1 (33), 119.1 (48), 101.0 (30)

19 a

Glucopyranosyl 21.47

951.2

403.1 (50), 371.1 (100), 223.1 (50)

dimethyloleoside isomer 2

20 a

453.2 (100), 421.2 (46), 393.1 (3), 371.2 (5), 299.1 (32), 22.92

Nuezhenide isomer 3

685.6 223.1 (14), 191.1 (8), 149.2 (11), 121.1 (13), 101.0 (15)

21 a

Methyloleoside23.44

925.3 (2), 855.3 (2), 771.4 (6), 701.3 (37), 539.3 (2), 469.2 1087.4

neonuezhenide

(2), 437.2 (1), 315.2 (12) 291.1 (100), 259.0 (5), 223.0 (3), 179.1 (10), 171.1 (13),

24.80

22

Ligstroside

523.5

2.9

153.1 (12), 139.1 (12), 127.1 (8), 101.0 (18) 23 a

26.19

Gl3 isomer 1

1071.8

24

26.64

Gl3

1071.8

685.3 (100), 453.2 (66), 421.1 (95) 839.3 (15), 685.3 (27), 523.1 (37), 453.1 (100), 421.3 (35), 299.2 (29)，223.3 (12)

25 a

27.77

Gl3 isomer 2

1071.8

26 a

29.24

Gl5 isomer

909.6

685.3 (31), 523.4 (31), 453.2 (100), 421.3 (39), 299.2 (23) 677.2 (14), 523.3 (9), 361.1 (27), 291.2 (100), 259.3 (46), 223.0 (4), 191.4 (4), 163.1 (4), 101.1 (4)

27 b

46.09

Oleanolic acid

455.3

407.2 (29)

455.3532

455.3520

2.6

28 b

46.09

Ursolic acid

455.3

407.2 (29)

455.3532

455.3520

2.6

a

Discovered for the first time in O. fragrans fruits. b Confirmed by comparison to authentic standards. c Determined by HPLC-ESI-QQQtrap. d

Determined by HPLC-ESI-Q Orbitrap.



Page 30 of 35

Table 2. Method Validation Using Seven Target Analytes Precision (%) Linear range Compound

Calibration curve

r (ng/mL)

(ng/mL)

Recovery

Repeat-

LOQ

Stability Intra-dayInter-day RSD(%) RSD(%)

ability

RSD (%) Mean

RSD (%)

(%)

RSD (%)

Salidroside

Y=161861X+7360

26.25-10500.00

0.9997

13.12

1.58

2.13

4.14

3.77

102.87 1.37

Acteoside

Y=578042X-4082

12.53-5010.00

1.0000

12.53

1.19

4.98

3.01

3.32

90.41

3.52

Isoacteoside

Y=499869X-14588

11.35-4540.00

0.9997

11.35

0.26

2.57

4.13

4.87

97.11

4.89

Neonuezhenide

Y=55319X-134

76.05-30420.00

1.0000

9.51

2.11

2.18

4.39

2.91

84.67

3.53

Nuezhenide

Y=59897X+7480

120.75-48300.00

1.0000

24.15

1.11

2.16

2.79

3.76

102.79 0.25

Oleanolic acid

Y=9176322X+86265

9.90-9900.00

0.9999

9.90

0.79

5.73

3.25

2.45

97.74

Ursolic acid

Y=7178054X+79344

9.76-9760.00

0.9997

9.76

0.58

8.90

2.11

4.61

104.50 4.10


3.22

Page 31 of 35


Table 3. Content Comparisons of Seven Compounds in Ten Batches of O. fragrans Fruits Batch 1a

Batch 2b

Batch 3b

Batch 4b

Batch 5b

Batch 6b

Batch 7a

Batch 8a

Batch 9b

Batch 10a

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

Salidroside

0.66

2.58

3.48

2.05

2.38

3.11

0.21

0.18

1.36

0.21

Acteoside

0.47

0.37

0.48

0.33

0.24

0.48

0.19

0.14

0.26

0.25

Isoacteoside

0.05

0.03

0.06

0.03

0.03

0.06

0.02

0.02

0.03

0.02

Neonuezhenide

0.61

0.92

0.55

0.55

0.81

0.46

0.82

0.48

0.81

0.21

Nuezhenide

11.78

16.99

16.87

13.76

15.77

26.31

6.34

6.07

18.33

12.44

Oleanolic acid

1.84

2.14

1.99

1.94

1.53

2.13

2.22

2.38

2.73

1.66

Ursolic acid

0.52

0.58

0.58

0.58

0.46

0.53

0.60

0.50

0.83

0.52

Compound

a

Cultivar belongs to O. fragrans Albus group. b Cultivar belongs to O. fragrans Luteus group.



Figure 1.


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Figure 2.



Figure 3.


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Table of Contents Graphic


Identification and Quantitation of the Bioactive ... - ACS Publications

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