Revista Brasileira de Farmacognosia (2021) 31:347–352
[Link]
SHORT COMMUNICATION
Simultaneous Determination of Flavonoids from Bamboo Leaf Extracts
Using Liquid Chromatography‑Tandem Mass Spectrometry
Xiabing Li1,2 · Wuqun Tao3 · Hang Xun2 · Xi Yao2 · Jin Wang2 · Jia Sun2 · Yongde Yue2 · Feng Tang2
Received: 19 November 2020 / Accepted: 3 May 2021 / Published online: 29 June 2021
© The Author(s) 2021
Abstract
An analytical method for the simultaneous determination of ten major functional flavonoids (isoorientin, orientin, vitexin,
isovitexin, apigenin, luteolin, tricin, quercetin, rutin, and kaempferol) in different bamboo species was developed by liquid
chromatography-tandem mass spectrometry. Chromatographic separation was carried out on a reversed-phase C-18 column
with acetonitrile and water as the mobile phases. Detection was performed in negative ion electrospray ionization mode
using multiple reaction monitoring mode. The correlation coefficients for the calibration curves ranged from 0.9955 to
0.9997. The limit of detection ranged from 1 to 45 ng/ml. The applicability of this analytical approach was confirmed by
the successful analysis of real leaf samples of four bamboo species, family Poaceae: Pleioblastus amarus (Keng) Keng f.,
Phyllostachys glauca McClure, Phyllostachys edullis (Carrière) [Link], and Indocalamus latifolius (Keng) McClure. The
total flavonoid contents were 3321.09, 3095.96, 4037.33, and 2808.42 mg/kg for P. amarus, P. glauca, P. edullis, and I.
latifolius, respectively.
Keywords Bamboo leaf flavonoids · LC–MS-MS · Multiple reaction monitoring · Different ethanol extracts
Introduction et al. 2012; Mao et al. 2013). Previous studies have reported
that flavonoid-rich bamboo leaf extracts have a variety of
Bamboo, an important building material and a potential biological effects including antioxidant, anticancer, antibac-
source of bioactive substances, is widely distributed in terial, antiviral, anti-inflammatory, and antimutagenic effects
the tropics and subtropics (Lu et al. 2005). Bamboo leaves (Lu et al. 2005; An et al. 2012; Van Hoyweghen et al. 2012).
have been used as food and folk medicine for more than These extracts can also be used as ingredients in dietary
1000 years in China (Lu et al. 2005; Gong et al. 2015). supplements and food additives (Lu et al. 2005; Zhang et al.
Numerous studies have shown that the major bioactive 2007; Koide et al. 2011; Gong et al. 2015), in part, due to
components of bamboo leaf extract are flavonoids including the antioxidant activity of bamboo leaves. Antioxidants from
kaempferol (1), rutin (2), orientin (4), isoorient (8), vitexin bamboo leaves can be added to the puffed grain food (corn,
(9) and isovitexin (10), and quercetin (5) (Zhang et al. 2005; rice, wheat, and amaranth, inter alia, for snacks and food
Lee et al. 2010; Wang et al. 2010; 2012; Wu et al. 2012; Ma product) meat products and oils, as authorized by the Min-
istry of Health, PR China (Lu et al. 2005). Bamboo leaf
flavonoids are also important in fat resistance and effective
* Feng Tang in the treatment of cancer and aging (Hu et al. 2000).
fengtang@[Link] Concerning the extensive utilization of bamboo leaf flavo-
1
School of Chemistry and Life Sciences, Chuxiong Normal noids in the functional food industry and commercial products,
University, Chuxiong 675000, Yunnan, China it is necessary to develop a method to quantitatively determine
2
National Forestry and Grassland Administration Key Open active flavonoids and ensure quality control of these extracts.
Laboratory, International Centre for Bamboo and Rattan, Some analytical methods have been reported for the simultane-
Beijing 100102, China ous quantitation of two or four flavonoid constituents, including
3
State Key Laboratory of Environmental Chemistry 8, 10, 4, and 9 in bamboo leaf extracts. Analytical methods
and Ecotoxicology, Research Center for Eco-Environmental include thin-layer chromatography (TLC) (Wang et al. 2012),
Sciences, Chinese Academy of Sciences, Beijing 100085, high-performance liquid chromatography HPLC (Zhang et al.
China
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348 Revista Brasileira de Farmacognosia (2021) 31:347–352
2005), high-performance capillary electrophoresis (HPCE) [Link], and Indocalamus latifolius (Keng) McClure, were
(Lu et al. 2005), high-performance thin-layer chromatography collected from Changning County, Sichuan Province,
(HPTLC) (Sun et al. 2010; Jian et al. 2011), and high-perfor- China in November, 2016 and identified by Guanghui Lai,
mance liquid chromatography-mass spectrometry (HPLC–MS) Guangde Forestry and Grassland Bureau (Anhui, China).
(Pereira et al. 2010). However, to date, liquid chromatography- Vouchers registered as CDBI0152311 and CDBI0152265
tandem mass spectrometry (LC–MS-MS) methods have not for P. amarus and I. latifolius were deposited in the her-
been developed for the simultaneous analysis of the ten major barium of Chengdu Institute of Biology, Chinese Acad-
flavonoids in bamboo leaves. LC–MS-MS has been recognized emy of Sciences. Vouchers registered as SM720601535
as a powerful analytical tool for the fast measurement of vari- and LBG00120870 for P. heterocycle and P. glauca were
ous analytes, such as polar, nonvolatile, high molecular mass deposited in the Chongqing Academy of Chinese Materia
compounds in natural product research compared to reversed- Medica herbarium and the Lushan Botanical Garden, Chi-
phase standalone HPLC coupled with diode array detection (Zu nese Academy of Sciences herbarium, respectively. Dried
et al. 2006). In this study, a simple, sensitive, and throughout bamboo leaf powder (10.0 g) was macerated in a mixture
LC–MS-MS method was first developed and validated for the (100 ml) of ethanol (EtOH)/H2O (3:2) and treated with a
simultaneous determination of ten flavonoids: kaempferol (1), sonicator (250 W, KQ-500, Kunshan Ultrasonic Instru-
rutin (2), tricin (3), orientin (4), quercetin (5), luteolin (6), api- ment Co., Ltd.) for 30 min at 50 °C. After repeated extrac-
genin (7), isoorientin (8), vitexin (9), and isovitexin (10), which tion (n = 3), the extracted solution obtained by filtration
are generally considered to be the major constituent flavonoids was concentrated under reduced pressure and then diluted
of bamboo leaves (Yang et al. 2017), in a single run and to with water to 50 ml. The extract, after extraction with
demonstrate the applicability of the method for the analysis of petroleum ether (3 × 150 ml), was evaporated to dryness
leaf samples from four species of bamboo. with a rotary evaporator and subjected to a macroporous
resin column (Diaion HP-20, Mitsubishi Chemical Corp.,
R1 Tokyo, Japan) at a flow rate of 10 ml/min using H 2O/
OH EtOH (100:0, 85:15, 70:30, 50:50, 30:70, 5:95 v/v) as the
R4 solvent to produce six extracts. The solvent was removed
HO O under reduced pressure. The residue was reconstituted in
R2
65% EtOH/H2O (10 ml), filtered through a 0.22-μm cel-
R3 R5 lulose membrane, transferred into glass vials, and stored
at − 20 °C until analysis.
OH O
LC–MS-MS analysis was conducted by using a Bruker
Advance UPLC system coupled with a Bruker EVOQ Elite
1 R1=R2=R3=R4=H; R5=OH triple quadrupole mass spectrometer (Bruker, Fremont,
2 R2=R3=R4=H; R1=OH; R5=OGlu6-1 Rha CA), which was connected to an electrospray ionization
3 R1=R2=OCH3; R3=R4=R5=H (ESI) source. The samples were injected onto an Agilent
4 R2=OH; R1=R3=R5=H; R4=Glu Eclipse XDB C-18 (4.6 × 150 mm, 5 μm) column. The
flow rate was 0.8 ml/min with a split ratio of 1:1, and
5 R2=R5=OH; R1=R3=R4=H
the column temperature was set at 40 °C. Each sample
6 R1=OH; R2=R3=R4=R5=H
was analyzed at least three times with an injection vol-
7 R1=R2=R3=R4=R5=H ume of 2 µl. The ESI source was operated in negative ion
8 R2=OH; R1=R4=R5=H; R3=Glu mode, and full scan mass spectral data were acquired over
9 R1=R2=R3=R5=H; R4=Glu a range from mass-to-charge (m/z) 50 to 800. Negative
10 R1=R2=R4 =R5=H; R3=Glu ESI mode was selected as the ionization mode for further
optimization experiments. Multiple reaction monitoring
(MRM) mode using the m/z transitions of the precursor
and product ions was applied to enhance sensitivity. The
optimized mass spectrometric parameters were as follows:
Materials and Methods nebulizer gas flow, 60 ml/min; probe gas flow, 50 ml/min;
cone temperature, 300 °C; cone gas flow, 20 ml/min; and
The ten flavonoid compounds were purchased from Shang- probe temperature, 300 °C.
hai Winherb Medical Technology Co., Ltd. (Shanghai, To estimate the analytical parameters of the methodology
China) with a purity above 98%. Leaves of four bamboo for bamboo samples, intra-day precision, limits of detec-
species, Pleioblastus amarus (Keng) Keng f., Phyllos- tion and quantification, matrix effects, and recovery were
tachys glauca McClure, Phyllostachys edullis (Carrière) evaluated. Five consecutive injections of each bamboo leaf
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Revista Brasileira de Farmacognosia (2021) 31:347–352 349
extracts were performed on the same day to evaluate the up with the procedures described above. The matrix effect
method’s precision. The results are expressed as relative was evaluated by comparing the slopes of the calibration
standard deviations (RSDs). Calibration curves were estab- curves obtained from the spiked bamboo leaf samples with
lished by injecting pooled standard solutions prepared from the slopes of the calibration curves obtained from the stand-
the standard mixtures. Calibration curves were constructed ards. The signal of the analyte was considered enhanced
by plotting the concentrations of each target analyte versus if the quotient of the spiked sample curve slope and the
the target analyte peak area using a linear regression analy- standard curve slope was higher than 1, whereas the signal
sis. A six-point calibration curve in the range of 0.1–50 µg/ of the analyte was considered suppressed if the value was
ml was generated with correlation coefficients (R2) between lower than 1.
0.9955 and 0.9997. The detection limits (LODs) and quan-
tification limits (LOQs) were estimated at signal-to-noise
(S/N) ratios of 3 and 10, respectively. Recovery tests were Results and Discussion
carried out (five replicates) by spiking the compounds at
three levels in the range of the calibration curves (1, 10, Individual target compound solutions were directly
and 50 µg/ml, final concentration added) in the bamboo injected into the ESI–MS-MS system to identify the
leaf samples before extraction, and a procedural blank was precursor and product ions under negative ionization
also carried out. Five replications of 10.0 g well-homoge- modes. After choosing the precursor ions and product
nized bamboo leaf composites were extracted and cleaned ions, the parameters for collision energy (CE) were
Fig. 1 The extracted ion chromatograms of ten flavonoids. a Refer- 0.35 µg/ml; luteolin (6, Rt = l8.53 min) 0.13 µg/ml; 0.0635 µg/
ence standards and b a mixture for one of the bamboo leaf extracts ml; apigenin (7, Rt = 20.97 min), 0.275 µg/ml; isoorientin (8,
(Pleioblastus amarus). Kaempferol (1, Rt = 21.18 min), rutin (2, Rt = 6.19 min) 1.5 µg/ml; vitexin (9, Rt = 10.98 min) 2.5 µg/ml; isovi-
Rt = 12.06 min) 0.85 µg/ml; tricin (3, Rt = 20.7 min) 0.0635 µg/ml; texin (10, Rt = 11.71 min) 6.5 µg/ml
orientin (4, Rt = 7.25 min) 5.25 µg/ml; quercetin (5, Rt = 19.07 min)
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350 Revista Brasileira de Farmacognosia (2021) 31:347–352
Table 1 Intra-day precision, Compound Linearity LOQ (ng/ml) LOD (ng/ml) Intra-day preci-
linearity, limits of detection sion (RSD %)
2
(LOD), and limits of Range (µg/ml) R
quantification (LOQ) of the
analytes with LC–MS/MS Apigenin 0.1–50 0.9993 3 1 1.12
Quercetin 0.1–50 0.9991 20 5 2.21
Rutin 0.1–50 0.9985 100 30 1.38
Luteolin 0.1–50 0.9971 5 2 3.12
Kaempferol 0.1–50 0.9987 5 2 2.34
Vitexin 0.1–50 0.9997 100 35 2.18
Isovitexin 0.1–50 0.9993 150 45 1.36
Orientin 0.1–50 0.9991 55 15 3.26
Isoorientin 0.1–50 0.9978 90 25 5.33
Tricin 0.1–50 0.9955 10 3 3.45
further optimized to obtain the maximum response of standard mixture after being optimized. Flavonoids were
the precursor and product ions (Table S1). In this study, eluted in the order of isoorientin (8, Rt = 6.19 min), ori-
different mobile phases, such as methanol (MeOH), ace- entin (4, Rt = 7.25 min), vitexin (9, Rt = 10.98 min), iso-
tonitrile (MeCN), and a mixture of MeOH and MeCN vitexin (10, Rt = 11.71 min), rutin (2, Rt = 12.06 min),
(50:50, v/v) with different compositions (ammonium luteolin (6, Rt = l8.53 min), quercetin (5, Rt = 19.07 min),
acetate, formic acid at various concentrations), were tricin (3, Rt = 20.7 min), apigenin (7, Rt = 20.97 min), and
tested. As expected, the addition of ammonium acetate kaempferol (1, Rt = 21.18 min).
into the mobile phases resulted in good peak shapes and Analytical parameters, such as the linearity, precision of
enhanced the detection sensitivity for the majority of the method, and LOD, were calculated for the developed
the target compounds. Finally, gradients consisting of LC–ESI–MS-MS method. The intra-day RSD values ranged
0.05% ammonium acetate in acetonitrile (mobile phase from 1.12 to 5.33% and were always < 10%, indicating good
A) and 0.05% ammonium acetate in water (mobile phase intra-assay variation (Table 1). For the linearity study, cali-
B) were chosen as the appropriate mobile phases for rou- bration curves were determined for all the individual ana-
tine LC–MS-MS analyses of the target compounds. The lytes in the six calibration standards (0.1–50 µg/ml) with
optimized gradient elution for LC separation was as fol- correlation coefficients ranging between 0.9955 and 0.9997
lows: 0 ~ 15.00 min, 15% A; 15.01 ~ 25.00 min, 35% A; (Table 1). The LOD and LOQ values for the target com-
25.00 ~ 27.00 min, 35% A ~ 100% A; 27.00 ~ 28.00 min, pounds ranged from 1 to 45 ng/ml and from 3 to 150 ng/
100% A ~ 15% A. The equilibration time before the next ml, respectively (Table 1). The recoveries of the bamboo
run was 6 min. Under the described chromatographic leaf flavonoids ranged from 68.45 to 112.31%, with standard
conditions, all the compounds were eluted in a total run deviations ranging from 2.3 to 9.1% for the 0.1, 0.2, and
time of 25 min. Figure 1 shows a chromatogram for all 0.4 µg/ml spiking tests (n = 5; Table S2). The results of the
Fig. 2 Total flavonoids in dif-
ferent ethanol fractions of four
bamboo leaf crude extracts
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Revista Brasileira de Farmacognosia (2021) 31:347–352 351
method validation revealed good sensitivity, precision, and Data Availability Some or all data, models, or code that support the
accuracy for the simultaneous determination of all of the findings of this study are available from the corresponding author upon
reasonable request.
target compounds by the developed method.
The developed LC–MS-MS method was finally applied
Declarations
to the determination of ten major flavonoids from four bam-
boo leaf extracts (P. amarus, I. latifolius, P. heterocycle, P. Conflict of Interest The authors declare no competing interests.
glauca) (Table S3) and the corresponding desorption solu-
tions (EtOH concentration: 0, 15, 30, 50, 70, and 95%, v/v) Open Access This article is licensed under a Creative Commons Attri-
of the four bamboo leaf extracts (Table S4, 5, 6, 7). As sum- bution 4.0 International License, which permits use, sharing, adapta-
marized in Table S3, all four bamboo leaf extracts contained tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
compounds 1–4, and the concentrations of these ten flavo- provide a link to the Creative Commons licence, and indicate if changes
noids were similar among the four species. Compounds 3, 4, were made. The images or other third party material in this article are
and 9 were identified as major components in all the bamboo included in the article’s Creative Commons licence, unless indicated
leaf extracts (Fig. S1), which is in agreement with previ- otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
ous studies (Jiao et al. 2007; Van Hoyweghen et al. 2012). permitted by statutory regulation or exceeds the permitted use, you will
The total concentrations of the flavonoids were 3321.09, need to obtain permission directly from the copyright holder. To view a
3095.96, 4037.33, and 2808.42 mg/kg in P. amarus, I. lati- copy of this licence, visit [Link]
folius, P. heterocycle, P. glauca respectively (Table S3) . The
most abundant flavonoids that were determined in the four
bamboo leaf extracts were 3, 2, and 4. It has been reported References
that these compounds are used as markers for the determi-
nation of bamboo leaf flavonoids in commercial products An F, Yang G, Tian J, Wang S (2012) Antioxidant effects of the orien-
(Jiao et al. 2007; Zhang et al. 2005). Fig. S1 shows the tin and vitexin in Trollius chinensis Bunge in D-galactose-aged
mice. Neural Regen Res 7:2565–2575. [Link]
compositions and contents of the ten flavonoids in different issn.1673-5374.2012.33.001
ethanol extracts of leaves from four species of bamboo. All Gong J, Xia D, Huang J, Ge Q, Mao J, Liu S, Zhang Y (2015) Func-
the target analytes were detected in only the 30% and 50% tional components of bamboo shavings and bamboo leaf extracts
EtOH extracts, and 2 and 4 were the dominant compounds and their antioxidant activities in vitro. J Med Food 18:453–459.
[Link]
in all of the examined samples. The highest content of total Hu C, Zhang Y, Kitts DD (2000) Evaluation of antioxidant and prooxi-
flavonoids was detected in the 50% EtOH extract (Fig. 2) in dant activities of bamboo Phyllostachys nigra var. henonis leaf
all of the examined samples. This finding can also provide extract in vitro. J Agric Food Chem 48:3170–3176. [Link]
information for the further purification of flavonoid-rich org/10.1021/jf0001637
Jian C, Yue Y, Feng T, Jin W (2011) HPTLC Analysis of the flavonoids
extracts. The compositions and distributions of the detected in eight species of Indocalamus leaves. JPC-J Planar Chromat
flavonoids were similar in the leaf extracts from the four 24:394–399. [Link]
species of bamboo (Fig. 2). The information obtained in Jiao J, Zhang Y, Liu C, Liu J, Wu X, Zhang Y (2007) Separation and
this study provides further support for the development of purification of tricin from an antioxidant product derived from
bamboo leaves. J Agric Food Chem 55:10086–92. [Link] oi.org/
bamboo leaf extract as food supplements. 10.1021/jf0716533
Koide CL, Collier AC, Berry MJ, Panee J (2011) The effect of bam-
Supplementary Information The online version contains supplemen- boo extract on hepatic biotransforming enzymes–findings from
tary material available at [Link] oi.o rg/1 0.1 007/s 43450-0 21-0 0158-1. an obese-diabetic mouse model. J Ethnopharmacol 133:37–45.
[Link]
Acknowlegements The helpful suggestions by the anonymous referees Lee HJ, Kim KA, Kang KD, Lee EH, Kim CY, Um BH, Jung SH
and editor-in-chief are gratefully acknowledged. (2010) The compound isolated from the leaves of Phyllostachys
nigra protects oxidative stress-induced retinal ganglion cells
Author Contribution Data curation: LXB; experimental investigation, death. Food Chem Toxicol 48:1721–1727
validation, and formal analysis: TWQ, XH, YX, WJ, and JS; funding Lu B, Wu X, Tie X, Zhang Y, Zhang Y (2005) Toxicology and safety
acquisition: TF and YYD; supervision: TF; original draft preparation: of anti-oxidant of bamboo leaves. Part 1: acute and subchronic
LXB; writing, review and editing: XH and TF. All the authors have toxicity studies on anti-oxidant of bamboo leaves. Food Chem
read and approved its submission. Toxicol 43:783–792. [Link]
Ma X, Yan R, Yu S, Lu Y, Li Z, Lu H (2012) Enzymatic acylation of
isoorientin and isovitexin from bamboo-leaf extracts with fatty
Funding This work was supported by The Basic Science Research
acids and antiradical activity of the acylated derivatives. J Agr
Fund Program of ICBR (No. 1632018007, 1632019022, 1632017005)
Food Chem 60:10844–10849. [Link]
and Yunnan Provincial Department of Education Science Research
Mao JW, Yin J, Ge Q, Jiang ZL, Gong JY (2013) In vitro antioxidant
Fund Project (No. 2020J0764).
activities of polysaccharides extracted from Moso Bamboo-Leaf.
Int J Biol Macromol 55:1–5. [Link]
2012.12.027
13
352 Revista Brasileira de Farmacognosia (2021) 31:347–352
Pereira CAM, Yariwake JH, McCullagh M (2010) Distinction of the Wu D, Chen J, Lu B, Xiong L, He Y, Zhang Y (2012) Application of
C-glycosylflavone isomer pairs orientin/isoorientin and vitexin/ near infrared spectroscopy for the rapid determination of antioxi-
isovitexin using HPLC-MS exact mass measurement and in- dant activity of bamboo leaf extract. Food Chem 135:2147–2156.
source CID. Phytochem Anal 16:295–301. [Link] [Link]
1002/pca.820 Yang JH, Choi MH, Yang SH, Cho SS, Park SJ, Shin HJ, Ki SH (2017)
Sun J, Yue Y, Tang F, Guo X (2010) Simultaneous HPTLC analysis Potent anti-inflammatory and antiadipogenic properties of bam-
of flavonoids in the leaves of three different species of bamboo. boo (Sasa coreana Nakai) leaves extract and its major constituent
JPC-J Planar Chromat 23:40–45. [Link] flavonoids. J Agric Food Chem 65:6665–6673. [Link]
2010.1.7 1021/acs.jafc.7b02203
Van Hoyweghen L, De Beer T, Deforce D, Heyerick A (2012) Phenolic Zhang Y, Bao B, Lu B, Ren Y, Tie X, Zhang Y (2005) Determination
compounds and anti-oxidant capacity of twelve morphologically of flavone C-glucosides in antioxidant of bamboo leaves (AOB)
heterogeneous bamboo species. Phytochem Anal 23:433–443. fortified foods by reversed-phase high-performance liquid chro-
[Link] matography with ultraviolet diode array detection. J Chromatogr
Wang J, Tang F, Yue Y, Guo X, Yao X (2010) Development and A 1065:177–185. [Link]
validation of an HPTLC method for simultaneous quantitation Zhang Y, Chen J, Zhang X, Wu X, Zhang Y (2007) Addition of anti-
of isoorientin, isovitexin, orientin, and vitexin in bamboo-leaf oxidant of bamboo leaves (AOB) effectively reduces acrylamide
flavonoids. J AOAC Int 93:1376–1383. [Link] formation in potato crisps and French fries. J Agric Food Chem
s00003-010-0634-7 55:523–528. [Link]
Wang J, Yue YD, Jiang H, Tang F (2012) Rapid screening for flavone Zu Y, Li C, Fu Y, Zhao C (2006) Simultaneous determination of cat-
C-glycosides in the leaves of different species of bamboo and echin, rutin, quercetin kaempferol and isorhamnetin in the extract
simultaneous quantitation of four marker compounds by HPLC- of sea buckthorn (Hippophae rhamnoides L.) leaves by RP-HPLC
UV/DAD. Int J Anal Chem 205101:1–8. [Link] with DAD. J Pharm Biomed Anal 41:714–719. [Link] rg/1 0.
2012/205101 1016/j.jpba.2005.04.052
13
