Introduction

 

Flavonoids are the important natural small molecule organic compounds, abundantly existing in plants. These are polyphenolic compounds from plant secondary metabolites (Wang and Yang 2016). Their structures are a series of C6-C3-C6 compounds with 2-phenylchromone as the basic mother nucleus (Tan 2002). Natural flavonoids are mostly derivatives of this basic structure and often exist in the form of glycosides in plants (Laggoune et al. 2011). Flavonoid glycosides are generally miscible with water, methanol, ethanol and other solvents, but difficult to dissolve in organic solvents such as ether, chloroform, benzene and so on (Kavita et al. 2018; Oluwaseun et al. 2018). The molecular structure of flavonoids is related to its biological activity, so various flavonoids have different pharmacological properties such as strong antioxidant, antibacterial, anti-inflammatory, anticancer and anti-aging (Cao et al. 2003; Roy et al. 2014; Zhang 2017).

Rhododendron pulchrum Sweet., a semi-evergreen shrub with bright colors and long flowering periods, has a high appreciation value and can be cultivated worldwide (Editorial Committee of Flora of China, Chinese Academy of Sciences 2004). A large number of research data have shown that wild Rhododendron are rich in flavonoids, and have many medicinal functions such as cough expectorant, antibacterial, anti-inflammatory, analgesic, etc. (Liu et al. 2010; Mittal et al. 2012; Sun et al. 2019). However, due to excessive picking, the resources of wild Rhododendron are getting lost gradually. In order to better protect such wild plant resources, we must find a plant source containing a lot of abundant flavonoids to replace wild Rhododendron. Therefore, the research team turned its attention to the R. pulchrum that can be cultivated everywhere. In the early stage of the project through preliminary analysis of the flavonoids in the R. pulchrum leaves, we find that it is an alternative resource for flavonoids with great potential and has high development and application value. Therefore, in this paper further analysis and research on the flavonoids of R. pulchrum has been reported.

At present, multi-level liquid chromatography / mass spectrometry technology has been widely used for the qualitative and quantitative analysis of natural compounds, such as the identification of compound fragments and the rapid analysis of unknown chemical components (Liu et al. 2017; Sun et al. 2018; Li et al. 2018). The research team has used HPLC-MS technology to analyze and identify 5 flavonoids in R. pulchrum leaves (Zhang et al. 2012). On this basis, HPLC-ESI-MS was used to identify the flavonoids and determine the content change of flavonoids in the flowering process in R. pulchrum, which will provide a theoretical basis for further application of flavonoids in R. pulchrum.

 

Materials and Methods

 

Experimental materials and treatments

 

From late march to late April 2019, the purple flowers were collected from the excellent R. pulchrum cultivated in Minjiang University. They were collected from four flowering stages, namely bud stage, initial flowering stage, full flowering stage, and late flowering stage. After picking in the morning, it was brought back to the laboratory and dried at 50°C. After crushing, it was passed through a 60-mesh sieve and collected for further analysis.

 

HPLC-MS analysis

 

Chromatographic conditions: Waters C18 column (4.6 mmΧ250 mm, 5 ΅m). Flow rate: 0.7 mL/min, initial column temperature is 35℃; injection volume is 20 ΅L, detection wavelength: 356 nm. Mobile phase A was 0.1% formic acid and mobile phase B was methanol. Elution procedure: 0 min, 31% B; 24 min, 43% B; 30 min, 50% B; 35 min, 60% B.

Mass spectrometry conditions: ion trap analyzer, electrospray ionization (ESI), negative ion detection mode, mass scan range (m/z): 200 to 700, capillary voltage: 3.5 kV, capillary outlet voltage: 100 V, drying gas temperature: 350℃, drying gas volume flow (N2): 10 L/min, atomizing gas pressure: 40 psi.

 

Standard curve drawing

 

Quercetin-3-galactoside, quercetin-3-O-arabinoside, quercetin-3-rhamnoside, and quercetin were measured by reversed-phase high-performance liquid chromatography under the above conditions, the peak area y is the ordinate, and the injection concentration x (μg/μL) is the abscissa. Linear regression analysis was performed. The standard curve equation for quercetin-3-galactoside is y = 3616171.61x-38716.31, r = 0.9998, and the linear range is 0.02~1.00 μg/μL. The standard curve equation of quercetin-3-O-arabinoside is y = 4046254.32x-36624.35, r = 0.9996, the linear range is 0.01~0.80 μg/μL. The standard curve equation of quercetin-3-rhamnoside is y = 4613441.22x-63649.51, r = 0.9995; the linear range was 0.02~1.00 μg/μL. The standard curve equation of quercetin is y = 3654072.63x-79893.53, r = 0.9995, the linear range is 0.01 ~ 0.80 μg/μL.

 

Fig. 1: TIC of the flavonoids in R. pulchrum flowers

 

 

Fig. 2: HPLC chromatogram of the flavonoids in R. pulchrum flowers

 

Preparation and determination of sample solutions

 

A 0.5 g of powder of R. pulchrum flowers was weighed accurately, then placed in a centrifuge tube and 80% ethanol solution was added to make the liquid material ratio of 120 mL/g. The mixture was put into the ultrasonic extraction system, and the extraction time was set at 60 min and the extraction temperature was 40°C. Then, the extract solution was centrifuged at a speed of 5000 r/min for 15 min (Shen et al. 2016). Finally, the supernatant was collected and passed through the 0.22 μm filter membrane to obtain the sample solution for analysis. The peak areas of quercetin-3-galactoside, quercetin-3-o-arabinoside, quercetin-3-rhamnoside and quercetin were calculated and substituted into the standard curve equation of the above reference, and the contents of each component were calculated.

 

Data analysis

 

Agilent Chem Station workstation and data processing software were used to analyze the total ion chromatogram and mass spectrometry data of each sample. The flavonoids in R. pulchrum flowers were identified by comparing the retention time of each component in R. pulchrum flowers, the data information of primary and secondary mass spectrometry, chemical composition database and related literature (Adam et al. 2004; Filip and Magda 2004; Li et al. 2009; Xu et al. 2010; Wu et al. 2011; Lv et al. 2015).

The content of each flavonoid component in the sample is determined by drawing a standard curve, the calculation formula was: "content of flavonoid component = (content of flavonoid component Χ total volume of flower sample extraction) Χ 100 / (quality of flower sample Χ injection volume)". Values indicating significant difference were analyzed for LSD and compared by the t test at P=0.05 between the bud stage and other flowering stages. The data are shown as mean ± standard deviation (SD) of three repetitions. All data were analyzed using SPSS 22.0 software.

 

Results

 

The separation of flavonoids in Rhododendron pulchrum flowers

 

HPLC-ESI-MS/Ms negative ion mode was used to analyze flavonoids in R. pulchrum flowers extracted with 80% ethanol. The (-) ESI-MS mass spectrometry total ion current (TIC) is shown in Fig. 1. The separation of flavonoids in R. pulchrum flowers were obtained by gradient elution with methanol solution as mobile phase (Fig. 2). Because the flavonoids R. pulchrum flowers are relatively polar and thermally unstable, ESI ion sources are used. In addition, the hydroxyl groups in the molecule easily form stable oxygen anions, so the total ion current (TIC) obtained by the analysis of negative ion mode has a better signal-to-noise ratio. The total ion chromatogram of the mass spectrum obtained is basically consistent with the UV chromatogram at 356 nm, but the baseline noise of the total ion chromatogram is large.

 

The identification of flavonoids in R. pulchrum flowers

 

HPLC-ESI-MS/Ms was used to analyze the molecular ion peaks in the chromatogram of flavonoids in R. pulchrum flowers by primary and secondary ion trap mass spectrometry, respectively. By comparing the corresponding ion peak information of flavonoids components in liquid chromatography with the information of the total ion current mass spectrum, and combining with the literature reports, the chemical structure of the six main peaks separated from flavonoids in Rhododendron pulchrum flowers by HPLC was deduced (Table 1).

 

Content of flavonoid components in R. pulchrum flowers at different flowering stages

 

Results showed that the content of flavonoids in R. pulchrum flowers decreased from bud stage to terminal flowering stage, especially from bud stage to initial flowering stage, indicating that the content of flavonoids reached a peak before flower opening (Table 2). The analysis of LSD showed there were the significant difference in the content of flavonoids between the bud stage and other flowering stages (Table 2). Among them, the content of quercetin-3-rhamnoside was 2.123 ± 0.081 mg/g at the flower bud stage, the content decreased by 27.65% (P<0.01) at the initial flowering stage, then decreased by 38.44% (P<0.01) at the full flowering stage, and finally decreased by 43.85% (P<0.01) at the end of the flowering period. However, there was no significant difference in the content of Quercetin-3-O-arabinoside between the bud stage and other flowering stages (P>0.05). The pigment of rhododendrons is mainly composed of flavonoids compounds. Thus, with the extension of the flower opening period, the color of flowers gradually fades, which may be related to the decrease in the content of flavonoids.

Table 1: Mass spectrometric analysis of flavonoids in R. pulchrum flowers

 

Peak No

Retention time (min)

MS (m/z)

MS2 (m/z)

Relative molecular mass

Compound

1

13.0

480.2 [M - H]-

330.4 [ (M - H)- 150] -

481.3

malva -3- arabinoside

2

20.4

463.3 [M - H]-

317.1 [ (M - H)- 146] -

464.4

myricetin 3- rhamnoside

3

22.3

463.2 [M - H]-

300.9 [ (M - H)- 162] -

464.4

quercetin -3- galactoside

4

27.0

433.2 [M - H] -

301.1 [ (M - H)- 132] -

434.4

quercetin -3-O-arabinoside

5

28.3

447.2 [M - H] -

301.0 [ (M - H)- 146] -

448.4

quercetin -3- rhamnoside

6

35.2

301.1 [M - H] -

301.1 [M - H] -

302.2

quercetin

 

Table 2: Content of flavonoid components in R. pulchrum flowers at different flowering stages

 

Flowering stage

Content of flavonoid components (mg/g)

quercetin -3- galactoside

Quercetin-3-O-arabinoside

Quercetin-3- rhamnoside

quercetin

Bud stage

1.435 ± 0.033

0.255 ± 0.023

2.123 ± 0.081

0.414 ± 0.053

Initial flowering stage

1.074 ± 0.027a

0.184 ± 0.011

1.536 ± 0.013a

0.285 ± 0.038b

Full flowering stage

0.881 ± 0.048 a

0.158 ± 0.025

1.307 ± 0.077a

0.263 ± 0.035b

Late flowering stage

0.815 ± 0.022 a

0.147 ± 0.043

1.192 ± 0.059a

0.253 ± 0.023b

Mean ± standard deviation. Different letters indicate significant difference according to T test at 0.05 level (a: P< 0.01, b: P < 0.05)

 

Discussion

 

The flower composition of Rhododendron is very complex. The flavonoids of Rhododendron flowers are mainly quercetin glycosides, malvidin glycoside and myricetin glycosides (Swiderski et al. 2004; Zhang et al. 2017). Quercetin glycosides are the most widespread flavonoids in Rhododendrons, followed by myricetin glycosides, which are auxiliary pigments of mallow glycosides (Li et al. 2008). HPLC-MS/MS technology was used to preliminarily identify six flavonoids in R. pulchrum flowers (Zhang et al. 2012; Lou et al. 2015). This result is consistent with the chromatographic data under the same separation conditions (Mok and Lee 2013). Mallow is a purplish red pigment in plants, and R. pulchrum are rich in mallow-3-arabinoside, which may be the main reason for the purple color of the flowers. The main reason of R. pulchrum flowers’ purple color may also be the co-color effect of flavonoids on anthocyanins, which stabilizes the dehydrogenation base of purple in the solution and prevents it from transforming into a colorless chalcone structure (Li et al. 2010; Oh et al. 2017).

Rhododendron not only has high appreciation value, but also high medicinal value. It is reported that flavonoids are widely distributed in Rhododendron plants, and they have many biological properties (Malkoc et al. 2016). This is the first report on antimicrobial activity of flavonoids of R. arboreum flowers (Sonar et al. 2012). The flavonoids in Rhododendron flower have antioxidant activities (Jung et al. 2007; Dede et al. 2019). Total favones of R. simsii Planch fower have a significant protective effect against cerebral ischemia-reperfusion injury (Chen et al. 2018). R. luteum is a great source of antioxidant and antitumor natural agents due to their capability of decreasing cancer cells proliferation (Demir et al. 2016). The results showed that the content of flavonoids was the highest in the flower bud period, and subsequently showed a significant decreasing trend. Therefore, in order to better develop and utilize the commercial utilization value of Rhododendron pigments and flavonoids, the flower bud period should be selected as the best harvesting time.

 

Conclusion

 

In this study, a rapid and sensitive HPLC-ESI-MS/MS method has been developed and was used to preliminarily identify six flavonoids in R. pulchrum flowers, which are malvacein-3-arabinoside, myricetin 3-rhamnoside, quercetin-3-galactoside, quercetin-3-O-arabinoside, quercetin-3-rhamnoside, and quercetin. This method was successfully applied to the determination of flavonoid content in R. pulchrum flowers at different flowering stages. In the future, the method can also be used as an efficient and reliable quality control method for other plant species.

 

Acknowledgements

 

The study was supported the Natural Science Foundation of Fujian Province (2018J01434), the Planned Project of Fujian Municipal Science and Technology Bureau (2019-g-54), National Undergraduate Innovation and Entrepreneurship Training Program (201910395002).

 

Author Contributions

 

MZ and QL planned the experiments, MZ, YZ and YH interpreted the results, MZ and BL made the write up and analyzed the data, YL made illustrations.

 

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