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.
References
Adam S, P Muras, H Koloczek (2004). Flavonoid
composition in frost-resistant Rhododendron
cultivars grown in Poland. Sci Hortic 100:139151
Cao WG, ZQ Liu, Y Shao, YZ Tao
(2003). A progress in pharmacological research of flavonoids.
Acta Bot Bor -Occidentalia Sin 23:22412247
Chen S, JH Zhang, YY Hu, DH Hu, ZW
Chen (2018). Total flavones of Rhododendron
simsii planch flower protect against cerebral
ischemia-reperfusion injury via the mechanism of cystathionine-γ-lyase-produced
H2S. Evi-based Compl Alt
2:111
Dede E, N Genc, M Elmastas, H Aksit, R Erenler (2019). Chemical constituents
isolated from Rhododendron ungernii with antioxidant profile. Nat Prod J 9:238243
Demir
S, I Turan, Y Aliyazicioglu
(2016). Selective cytotoxic effect of Rhododendron luteum extract on human colon and liver cancer
cells. J Buon
21:883888
Editorial Committee of
Flora of China, Chinese Academy of Sciences (2004).
Flora of China, Vol 57, p: 384.
Science Press, Beijing, China
Filip C, C Magda (2004), Mass
spectrometry in the structural analysis of flavonoids. J Mass Spectrom 39:115
Jung SJ, DY Kim, YH Hong, JH Lee, HN
Song, YD Rho, NI Back (2007). Flavonoids from the flower of Rhododendron yedoense
var. poukhanense
and their antioxidant activities. Arch
Pharm Res 30:146150
Kavita
S, M Neelima, RL Yong (2018). Extraction,
characterization and biological activity of citrus flavonoids. Rev Chem Eng
39:9871001
Laggoune
S, I Brouard, F Leon, CA Calliste,
JL Duroux, J Bermejo (2011). Lignans
and an abundant flavone glycoside with free-radical scavenging activity from
the roots of the endemic species Stachys mialhesi de noι. Rec Nat Prod 5:237241
Li C H, LS Wang, QY Shu, YJ Xu, J
Zhang (2008). Pigment composition of petals and floral color
during the blooming period in Rhododendron
mucronulatum. Acta Hortic Sin 35:10231030
Li J, K Jiang, LJ Wang, G Yin (2018).
HPLC-MS/MS determination of flavonoids in Gleditsiae spina for its quality assessment. J Sep Sci 41:17521763
Li X F, HZ Jin, G Chen, M Yanf, Y Zhu, YH Shen (2009). Flavonoids
from the aerial parts of Rhododendron primulaeflorum. Nat Prod Res Dev 4:612615
Li Y, CL Zhao, XN Yang, HR Li, YQ
Zhou, L Su (2010). Research advances in the relationship between the molecular
structures of anthocyanins and their stability and colorations. J Yunnan Agric Univ 25:712720
Liu, JH, YY Cheng, CH Hsieh, TH Tsai
(2017). Identification of a multicomponent traditional herbal
medicine by HPLCMS and electron and light microscopy. Molecules 22:22422247
Liu, YZ, YG Cao, JQ Ye, WG Wang, KJ
Song, XL Wang, CH Wang, RT Li, XM Deng (2010). Immunomodulatory effects of proanthocyanidin
A-1 derived in vitro from Rhododendron spiciferum. Fitoterapia 81:108114
Lou
XW, QH Lin, GY Zhang, WY Liu, F Feng, W Qu (2015). Identification
and characterization of three new flavonoids from Rhododendron dauricum. Chin J Nat Med 13:628633
Lv
HH, X Wang, Y He, H Wang, Y Suo (2015). Identification and quantification of
flavonoid aglycones in rape bee pollen from Qinghai-Tibetan plateau by
HPLC-DAD-APCI/MS. J Food Copmos Anal 38:4954
Malkoc
M, AQ Laghari, S Kolayli, Z Can (2016). Phenolic
composition and antioxidant properties of Rhododendron
ponticum: Traditional nectar source for mad
honey. Anal Chem Lett 6:224231
Mittal, AK, A Kaler, UC Banerjee (2012).
Free radical scavenging and antioxidant activity of silver nanoparticles
synthesized from flower extract of Rhododendron
dauricum. Nano
Biomed Eng 4:118124
Mok SY, S Lee (2013). Identification of flavonoids and flavonoid rhamnosides
from Rhododendron mucronulatum
for. albiflorum and their inhibitory activities against
aldose reductase. Food Chem
136:969974
Oh SM, JH Chun,
MK Lee, JB Kim, SJ Kim (2017). Simultaneous analysis of anthocyanins and flavonols in
various flower colors of Rhododendron schlippenbachii (Royal Azalea). Kor J Agric Sci
44:104113
Oluwaseun RA, HA Nour, AO Olusegun
(2018). Optimization of
microwave-assisted extraction of flavonoids and antioxidants from vernonia amygdalina leaf using
response surface methodology. Food
Bioprod Process 107:3648
Roy
JD, AK Handique, CC Barua, A Talukdar, FA Ahmed (2014). Evaluation of phytoconstituents
and assessment of adaptogenic activity in vivo in various extracts of Rhododendron arboreum
(leaves). Cancer Res 73:75157522
Shen
TB, M Zhang, XY Tang, ZX Li (2016). Optimization of
extraction technology of anthocyanins from Rhododendron
pulchrum Sweet. flowers
based on uniform design. J Jinggangshan Univ 37:2428
Sonar PK, R Singh, S Khan, SK Saraf (2012).
Isolation, characterization and activity of the flowers of Rhododendron arboreum
(Ericaceae). E-J Chem
9:631636
Sun, MJ, YW Yin, J Wei, XP Chen, HZ
Ouyang, YX Chang, XM Gao, J He (2018). Development and validation of a
HPLC-MS/MS method for simultaneous determination of twelve bioactive compounds
in Epimedium:
Application to a pharmacokinetic study in rats. Molecules 23:13221326
Sun N, Y Qiu,
Y Zhu, JJ Liu, HQ Zhang, QH Zhang, MK Zhang, GJ Zhang, C Zhang, GM Yao (2019). Rhodomicranosides A-I, analgesic
diterpene glucosides with diverse carbon skeletons from Rhododendron micranthum. Phytochemistry 158:112
Swiderski
A, P Muras, H Koloczek
(2004). Flavonoid composition in frost-resistant Rhododendron cultivars grown in Poland.
Sci Hortic
100:139151
Tan RX (2002). Analysis of Plant Components, pp:486502.
Science Press, Beijing, China
Wang HB, JF Yang (2016). Natural Product Chemistry, pp:7485. Chemical Industry Press, Beijing, China
Wu LY, XD Luo, LF Dai, JF Cao, LF
Liu, HY Hong, WY Pan (2011). Extraction and primary
identification of anthocyanidins in Rhododendron
flowers. Food Sci 23:1922
Xu FQ, HB Liu, JG Luo, JL Zhang, HS
Gua (2010). Studies on the chemical
constituents and meridian doctrine of Polygonum
aviculare. J Ocean Univ China 40:101104
Zhang M, DR Pan, YF Zhou, QQ Zhu,
SM Wang (2012). Analysis of the flavonoids in the leaves of Rhododendron pulchrum
Sweet. by HPLC-MS. Med Plant 3:2124
Zhang Q (2017). Anticancer effects
of flavonoids and flavonols, pp:1437.
Chemical Industry Press, Beijing, China
Zhang
XZ, B Zhao, HM Zeng, HF Shen, JJ Xu (2017). Comparative analysis of composition
and content of pigments in petals of three different colors of Rhododendron calophytum
in Qinling mountains. J Northwest For
Univ 32:6268