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Analysis of Flavonoids in Lotus (Nelumbo nucifera) Leaves and Their Antioxidant Activity Using Macroporous Resin Chromatography Coupled with LC-MS/MS and Antioxidant Biochemical Assays


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Lotus (Nelumbo nucifera) leaves, a traditional Chinese medicinal herb, are rich in flavonoids. In an effort to thoroughly analyze their flavonoid components, macroporous resin chromatography coupled with HPLC-MS/MS was employed to simultaneously enrich and identify flavonoids from lotus leaves. Flavonoids extracted from lotus leaves were selectively enriched in the macroporous resin column, eluted subsequently as fraction II, and successively subjected to analysis with the HPLC-MS/MS and bioactivity assays. Altogether, fourteen flavonoids were identified, four of which were identified from lotus leaves for the first time, including quercetin 3-O-rhamnopyranosyl-(1→2)-glucopyranoside, quercetin 3-O-arabinoside, diosmetin 7-O-hexose, and isorhamnetin 3-O-arabino- pyranosyl-(1→2)-glucopyranoside. Further bioactivity assays revealed that these flavonoids from lotus leaves possess strong antioxidant activity, and demonstrate very good potential to be explored as food supplements or even pharmaceutical products to improve human health.
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Molecules 2015, 20, 10553-10565; doi:10.3390/molecules200610553
ISSN 1420-3049
Analysis of Flavonoids in Lotus (Nelumbo nucifera) Leaves and
Their Antioxidant Activity Using Macroporous Resin
Chromatography Coupled with LC-MS/MS and Antioxidant
Biochemical Assays
Ming-Zhi Zhu 1, Wei Wu 2, Li-Li Jiao 2, Ping-Fang Yang 1 and Ming-Quan Guo 1,*
1 Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,
Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China;
E-Mails: (M.-Z.Z.); (P.-F.Y.)
2 Changchun University of Chinese Traditional Medicine, Changchun 130000, China;
E-Mails: (W.W.); (L.-L.J.)
* Author to whom correspondence should be addressed; E-Mail:;
Tel./Fax: +86-27-8751-8018.
Academic Editor: Isabel C. F. R. Ferreira
Received: 27 March 2015 / Accepted: 29 May 2015 / Published: 8 June 2015
Abstract: Lotus (Nelumbo nucifera) leaves, a traditional Chinese medicinal herb, are rich
in flavonoids. In an effort to thoroughly analyze their flavonoid components, macroporous
resin chromatography coupled with HPLC-MS/MS was employed to simultaneously enrich
and identify flavonoids from lotus leaves. Flavonoids extracted from lotus leaves were
selectively enriched in the macroporous resin column, eluted subsequently as fraction II,
and successively subjected to analysis with the HPLC-MS/MS and bioactivity assays.
Altogether, fourteen flavonoids were identified, four of which were identified from lotus
leaves for the first time, including quercetin 3-O-rhamnopyranosyl-(12)-glucopyranoside,
quercetin 3-O-arabinoside, diosmetin 7-O-hexose, and isorhamnetin 3-O-arabino-
pyranosyl-(12)-glucopyranoside. Further bioactivity assays revealed that these flavonoids
from lotus leaves possess strong antioxidant activity, and demonstrate very good potential to
be explored as food supplements or even pharmaceutical products to improve human health.
Keywords: lotus leaves; antioxidant activity; macroporous resin chromatography;
mass spectrometry
Molecules 2015, 20 10554
1. Introduction
Lotus (Nelumbo nucifera), a common perennial aquatic herb, is extensively cultivated in eastern Asia,
particularly in China [1]. All parts of lotus, including the leaves, stamens, flowers, seeds and rhizomes,
have been used as traditional Chinese medicines or vegetables for thousands of years [2]. The leaves of
lotus are traditionally used for the treatment of haematemesis, haematuria, metrorrhagia, hyperlipidaemia,
fever and inflammatory skin conditions [3]. In recent years, the antioxidant [4], antiviral [5], anti-obesity [6]
and lipolytic activities [7] of lotus leaves have been reported and attracted more and more interest, while
phytochemicals from lotus leaves and their associated potential activities have not yet been fully
explored. The annual production of lotus leaves now exceeds 800,000 tons in China, but most of them
are discarded as agricultural wastes by farmers [8]. It is thus highly desirable to expedite the research on
how to make the best use of lotus leaves as potential products for the food or pharmaceutical industries.
In this regard, it is of primary importance to conduct a thorough analysis of the major chemical
components from lotus leaves and their associated bioactivities.
It is reported that the lotus leaves are rich in flavonoids [5] and alkaloids [9], and several flavonoids
have been isolated. Kashiwada et al. isolated and identified five flavonoid glycosides (quercetin 3-O-β-
D-glucuronide, quercetin 3-O-β-D-xylopyranosyl-(12)-β-D-galactopyranoside, rutin, isoquecitrin and
hyperin) by nuclear magnetic resonance spectroscopy (NMR) [5]. Ohkoshi et al. also identified eight
flavonoids (quercetin 3-O-α-arabinopyranosyl-(12)-β-D-galactopyranoside, rutin, (+)-catechin,
hyperoside, isoquercitrin, quercetin, and astragalin) [7]. Till now, ten flavonoids were isolated from lotus
leaves [5,7,10]. Flavonoids, a type of phenolic compounds, have attracted extensive attention because
of their strong antioxidant activity and their ability to reduce the formation of free radicals and to
scavenge free radicals [11–13]. It is considered that oxidative damage is attributable to excess active
oxygen species generated in the body [14], and the antioxidant defense system plays an important role
in human health [15]. In order to reduce damage to the human body and prolong the storage stability of
foods, synthetic antioxidants are often used in these processes [16]. However, the use of synthetic
antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), has been
questioned for the possibility of being carcinogenic and causing liver damage [17]. Compared with
synthetic antioxidants, antioxidants from natural sources are characterized by their less toxicity, and
better health effects. Thus, the focuses of this study were put on the flavonoids from lotus leaves and
their associated antioxidant activity.
So far, several methods for the enrichment and separation of flavonoids in lotus leaves including
liquid-liquid extraction [10], SPE [18], high-speed counter-current chromatography (HSCCC) [19] have
been developed. However, these methods have several limitations, such as low capacity, low yields, or
the need for special instrumentation. Furthermore, these methods share similar difficulties in completely
separating flavonoids and alkaloids, which are the major active ingredients of lotus leaves.
Comparatively, macroporous resin chromatography, with its properties of high adsorption capacity,
good stability, low operational cost, and simple procedure, is one of the most efficient methods to
separate bioactive components from crude herbal material extracts [20]. Nowadays macroporous resins
chromatography has been successfully applied in industry for separation and preparation of flavonoids,
glycosides, saponins, and so on [21]. In this context, we strove to develop an off-line two-dimensional
chromatography method combining macroporous resin and reverse phase liquid chromatography to
Molecules 2015, 20 10555
firstly enrich the flavonoids from the crude extracts of lotus leaves in the first dimension, then conduct
the subsequent chemical and activity analysis of the flavonoid components.
2. Results and Discussion
2.1. Analysis of Flavonoids by HPLC-MS/MS
The highest total flavonoid content (TFC) was determined in fraction II (690.5 mg isoquercetin
equivalents/g sample, Table 1), while very few peaks were detected in fractions I, III and IV in
chromatographic profiles recorded at 350 nm, so fractions I, III and IV were thus deemed to have very
few or even no flavonoids. A successful enrichment and separation of total flavonoids from lotus leaves
was thus achieved by the D101 macroporous resin chromatography, and the resultant flavonoids were
then ready for the subsequent chemical and bioactivity analysis. After the flavonoids from the lotus leaves
extracts were successfully enriched in fraction II using the D101 macroporous resin chromatography,
HPLC-MS/MS was then employed to identify those flavonoids. Figure 1 shows the chromatographic
profile of fraction II at 350 nm, where fourteen peaks were well detected and resolved, representing at least
14 lotus leaves flavonoids. To identify these already resolved flavonoids, LC-MS/MS experiments were
conducted. The LC and MS/MS data, including retention times, molecular ions, aglycone ions and some
important fragment ions are listed in Table 1. The fragmentation of O-glycosyl flavonoids in the negative
ion mode (NI) is characterized by the loss of the sugar moieties, and deprotonated aglycone species (Y0)
or radical aglycone ion ([Y0 H].) fragments are obtained [22,23]. The MS/MS model of flavonoid
O-glycosides is shown in Figure 2. For flavonol mono-O-glycosides, glycosylation took place at the
3-position if the relative abundance of the [Y0 H]. ion was significantly higher than that of the Y0 ion,
and the situation is reversed when glycosylation happens at the 7-position [22]. Till now, five aglycones,
kaempferol, quercetin, isorhamnetin, myricetin and diosmetin, were identified from lotus [24].
Figure 1. The HPLC profile of flavonoids in fraction II from lotus leaves recorded at 350 nm.
Figure 2. The MS/MS model of flavonoid O-glycosides.
Molecules 2015, 20 10556
As listed in Table 1, peak 1 showed a [M H] ion at m/z 479, with its [Y0 H]. ion at m/z 316 (loss
of a hexose moiety), indicating that it was myricetin monohexoside, and the glycosylation took place at
the 3-position based on the characteristic [Y0 H]. fragment ion. Peak 1 was therefore identified as
myricetin 3-O-hexose, which has been previously found in lotus leaves and flowers [24]. Similar to the
MS/MS model of peak 1, the presence of a [M H] ion at m/z 477 and the corresponding [Y0 H].
ion at m/z 314 for peak 13 indicated that it was an isorhamnetin monohexoside identified as isorhamnetin
3-O-hexose, which has been previously reported [18]. Peak 2 exhibited the [M H] ion at m/z 595 with
its [Y0 H]. ion at m/z 300 (loss of a pentose and a hexose moiety), indicating that it was a quercetin
diglycoside. For flavonol O-diglycosides, the mass spectrometric behaviors of diglycosides are notably
different depending on the linkage between the two monosaccharides. A [Y0 H]. ion tends to be
generated in the case of a C1C2 linkage between the two monosaccharides, while the Y0 ion is
indicative of a C1C6 linkage [22]. The higher abundance of [Y0 H]. ion at m/z 300 for peak 3
indicated that the interglycosidic linkage between the two monosaccharides in this compound was
C1C2, and peak 2 was thus identified as quercetin 3-O-arabinopyranosyl-(12)-galactopyranoside,
which has been reported in lotus leaves [25].
Table 1. Identification of flavonoids in fraction II of lotus leaves by LC-MS/MS.
Peak No. Rt (min) a NI-MS MS/MS Identification
1 25.9 479 316 Myricetin 3-O-hexose
2 27.4 595 300 Quercetin 3-O-arabinopyranosyl-(12)-galactopyranoside
3 33.0 609 300 Quercetin 3-O-rhamnopyranosyl-(12)-glucopyranoside
4 34.1 463 300 Quercetin 3-O-galactoside (hyperoside)
5 35.5 463 300 Quercetin 3-O-glucoside (isoquercitrin)
6 37.2 477 301 Quercetin 3-O-glucuronide
7 38.7 433 300 Quercetin 3-O-arabinoside
8 40.6 447 284 Kaempferol 3-O-galactoside
9 43.7 447 284 Kaempferol 3-O-glucoside (astragalin)
10 45.7 461 285 Kaempferol 3-O-glucuronide
11 47.5 461 446; 298; 283 Diosmetin 7-O-hexose
12 52.6 609 314; 299 Isorhamnetin 3-O-arabinopyranosyl-(12)-glucopyranoside
13 56.7 477 314 Isorhamnetin 3-O-hexose
14 57.9 491 315 Isorhamnetin 3-O-glucuronide
a Rt: retention time on HPLC.
As shown in Figure 3A,D, peaks 3 and 12 exhibited the same [M H] ions at m/z 609 with
[Y0 H]· ions at m/z 300 and 314, indicating that they were quercetin diglycoside and isorhamnetin
diglycoside, respectively. Considering the similar MS/MS spectrometric behaviors with peak 2, peaks 3
and 12 were identified as quercetin 3-O-rhamnopyranosyl-(12)-glucopyranoside and isorhamnetin
3-O-arabinopyranosyl-(12)-glucopyranoside. These two flavonoids were identified in this study for
the first time. In addition, the presence of the fragment ion at m/z 299 ([Y0 H CH3]·) of peak 12
indicated that the methoxyflavone readily lost a methyl group [26].
Molecules 2015, 20 10557
Figure 3. The MS/MS spectra of newly identified flavonoids in fraction II from lotus leaves:
(A) peak 3; (B) peak 7; (C) peak 11; (D) peak 12.
Peaks 4 and 5 exhibited the same [M H] ions at m/z 463 with [Y0 H]. ions at m/z 300 (loss of a
hexose moiety), indicating that they are quercetin monohexoside isomers with a hexose conjugated at
the 3-position. In addition, peak 4 had a shorter retention time than peak 5. Considering that glycosides
linked with galactose elute before glucose linkages [27,28], peaks 4 and 5 were identified as quercetin
3-O-galactoside (hyperoside) and quercetin 3-O-glucoside (isoquercitrin) [25], and the results were
further validated by comparing their LC-MS with the corresponding standards.
Peak 6 showed a [M H] ion at m/z 477, with a Y0 ion at m/z 301 (loss of a glucuronic acid).
For glucuronic acid glycosides, only the Y0 ion was observed during the MS/MS process, and the
glucuronic acid glycoside was conjugated at the 3-position. Thus, peak 6 was identified as quercetin
3-O-glucuronide, which has previously been reported to be the major flavonoid in lotus leaves [25]. Like
the MS/MS spectrometric behavior of peak 6, the ions at m/z 461 ([M H]), m/z 285 (Y0), 491
([M H]) and m/z 315 (Y0) for peaks 10 and 14 indicated that they were kaempferol and isorhamnetin
glucuronic acid glycosides, respectively. Thus, peaks 10 and 14 were identified as kaempferol
3-O-glucuronide and isorhamnetin 3-O-glucuronide [24,29].
Molecules 2015, 20 10558
As shown in Figure 3B, peak 7 showed a [M H] ion at m/z 433, with a [Y0 H]. ion at m/z 300
(loss of a pentose moiety), indicating that it was quercetin monopentoside, and glycosylation took place
at the 3-position. Peak 7 was therefore identified as quercetin 3-O-arabinoside. Its MS/MS spectra agreed
with a compound identified in Juglans regia L. leaves [30], while it has been found in lotus leaves in
this work for the first time.
For peaks 8 and 9, the same [M H] ions were observed at m/z 447, with their [Y0 H]. ions at m/z
284 (loss of a hexose moiety), indicated that both were kaempferol 3-O-hexoses. Furthermore, peak 8
had a shorter retention time than that of peak 9. Thus, peaks 8 and 9 were identified as kaempferol
3-O-galactoside and kaempferol 3-O-glucoside (astragalin), which have previously been reported in lotus
petals [29]. In addition, peak 9 was further confirmed by comparing the LC-MS/MS spectra with the
corresponding standard.
As shown in Figure 3C, peak 11 showed a [M H] ion at m/z 461, with a [Y0 H]. ion at m/z 298
(loss of a hexose moiety), indicating that it was diosmetin monohexoside, and glycosylation took place
at the 7-position. The presence of the ions at m/z 446 ([M – CH3]) and 283 ([Y0 – H CH3].) indicated
that the aglycone of diosmetin easily lost a methyl group. Peak 11 was identified as diosmetin 7-O-hexose,
which has also been found in lotus leaves for the first time.
2.2. Antioxidant Activity of Flavonoids from Lotus Leaves
The antioxidant activity of plant extracts cannot be evaluated by only one single method due to the
complex nature of phytochemicals, and antioxidant activity determination is highly reaction-mechanism
dependent [12]. Multiple chemical or biological assays have been developed to evaluate the antioxidant
activity and explain the antioxidant mechanism of action of plant extracts. Of those, the DPPH assay,
ABTS assay and reducing power assay are the most commonly used assays to evaluate the antioxidant
activities of plant extracts [31]. In view of this, a series of assays including DPPH scavenging activity,
ABTS scavenging activity and FRAP were used for the determination of the antioxidant activity of the
fraction of lotus leaves. Results of these evaluations were expressed as Trolox equivalents and IC50
values, which are shown in Table 2. Fraction II showed good free radical scavenging activity in the
DPPH assay. The activity of fraction II (4695.3 μmol·TE/g) was higher than that of BHT (3612.3 μmol·TE/g,
positive control). In terms of IC50, fraction II (IC50 = 0.101 mg/mL) had a lower value than BHT
(IC50 = 0.121 mg/mL), which implied that fraction II possessed a stronger radical scavenging activity
in the DPPH assay. For scavenging activity pattern in the ABTS assay, the activity of fraction II
(5012.3 μmol·TE/g) was higher than that of BHT (4567.0 μmol·TE/g), while the IC50 value of fraction
II (IC50 = 0.138 mg/mL) was lower than that of BHT (IC50 = 0.143 mg/mL). With regard to the ferric
reducing capacity of fraction II, the trend was almost the same as that of the DPPH and ABTS assay.
The reducing power activity of fraction II (500.5 mmol Fe2+/100 g) was nearly the same with that of
Trolox (642.1 mmol Fe2+/100 g).
Molecules 2015, 20 10559
Table 2. Total flavonoids in fraction II from lotus leaves a and their corresponding
antioxidant activity.
Sample Total Flavonoid
(mg IE/g)
(mmol Fe2+/100 g)
(μmol TE/g) IC50 value b
(mg/mL) (μmol TE/g) IC50 value b
Fraction II 690.5 ± 35.8 4695.3 ± 144.3 0.101 ± 0.007 5012.3 ± 133.8 0.138 ± 0.007 500.5 ± 62.8
BHT nt 3612.3 ± 170.9 0.121 ± 0.004 4567.0 ± 155.6 0.143 ± 0.004 nt
Trolox nt nt 0.112 ± 0.005 nt 0.119 ± 0.005 642.1 ± 55.7
a Each value is presented as the mean ± SD of three replicate determinations; b IC50 value was determined to
be the effective concentration at which DPPH and ABTS radicals were inhibited by 50%, respectively. IE,
isoquercetin equivalents; TE, Trolox equivalents; nt, not tested.
Taken together, the above results demonstrate that fraction II from lotus leaves possesses good
antioxidant potential, and the antioxidant activity of fraction II was nearly the same as that of BHT. This
capability may be correlated to the different flavonoid components identified in the lotus leaves, whose
activity has been relative to the electron donating ability associated with the degree and position of
hydroxylation and methoxylation on the B-ring [32]. The flavonoids possessing a catecholic B-ring,
such as hyperoside (4) isoquercitrin (5) and quercetin 3-O-arabinoside (7) (which also possess a double
bond at the 2 position of the C ring, conjugated with the 4-oxo group), are probably the derivatives that
contribute the most to the total antioxidant activity, because the presence of a catechol moiety confers
greater stability to the aroxyl radicals formed upon reaction with radical compounds. Kaempferol
3-O-galactoside (8) and astragalin (9), which only possess a double bond at the 2 position of the C ring
(again conjugated with the 4-oxo function group) and a phenolic B-ring, probably contribute to the total
antioxidant activity to a lesser extent than those of the compounds mentioned above.
3. Experimental Section
3.1. Chemicals and Materials
Three flavonoid glycoside standards (quercetin 3-O-galactoside (hyperoside), quercetin 3-O-glucoside
(isoquercitrin), kaempferol 3-O-glucoside (astragalin)) were purchased from Shanghai Tauto Biotech
(Shanghai, China). HPLC-grade solvents (acetonitrile and formic acid), butylated hydroxytoluene
(BHT), 1,3,5-tri(2-pyridyl)-2.4.6-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic
acid (Trolox), α,α-diphenyl-β-picrylhydrazyl (DPPH), and 2,2-azinobis-(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS) were purchased from Sigma-Aldrich Corp. (Shanghai, China). HPLC-grade water
was obtained using a Milli-Q System (Millipore, Billerica, MA, USA). Other chemicals of analytical
grade were obtained from Shanghai Chemical Reagent Corp. (Shanghai, China). Millipore membranes
(0.22 μm) were purchased from Jinteng Experiment Equipment Corp. (Tianjin, China). D101 macroporous
resin was purchased from an industrial chemical company affiliated with Nan Kai University (Tianjin,
China). Fresh lotus leaves were collected from Guangchang (Jiangxi, China) in 2013 and stored at
40 °C. Lotus leaves were dried at 45 °C, and then stored at 4 °C before use.
Molecules 2015, 20 10560
3.2. Extraction and Fractionation of Crude Lotus Leaves Extracts
The dried lotus leaves were powdered (100 g) and ultrasonically extracted at room temperature for
40 min with 1000 mL of 70% ethanol. After three extractions, the extracts were combined and filtered,
and the supernatants were evaporated under reduced pressure and lyophilized to afford dark-green
residues which were dissolved in H2O (50 mL) and were subjected to liquid/liquid partitioning with
petroleum ether (B.P. 60–90 °C) in order to remove chlorophyll. Later the lower layer was evaporated,
and fractionated by the D101 macroporous resin chromatography. The column was washed with distilled
water to remove water soluble impurities (sugar, protein, and other water-soluble molecules) and then
eluted successively with 30, 50, 70, and 90% ethanol and named fractions I, II, III and IV, respectively.
The weights of crude extracts and fractions (I–IV) were 27.6 g, 3.1 g, 3.7 g, 1.0 g and 1.1 g, respectively.
Aliquots of the eluents were then subjected to further analysis.
3.3. Determinations of Total Flavonoids Content (TFC)
Total flavonoids content was determined using a previously described colorimetric method [33].
Briefly, a 30 μL aliquot of appropriately diluted sample solution was mixed with 180 μL of distilled
water in a well of a 96-well plate and 10 μL of a 5% NaNO2 solution was added subsequently. After
6 min, 20 μL of 10% AlCl3 solution was added and allowed to stand for 6 min before an addition of
60 μL of 4% NaOH solution. The absorbance of the mixture was determined at 510 nm vs. a water blank
using a multifunctional microplate reader (Infinite M200 PRO, Tecan, Männedorf, Switzerland) after
15 min. Isoquercitrin was used as standard compound for the quantification of total flavonoids. All
values were expressed as milligrams of isoquercitrin equivalents per gram of sample (mg IE/g sample).
3.4. HPLC Analysis of Flavonoids
The analysis of flavonoids was carried out using a Thermo Accela 1250 U-HPLC system (Thermo
Fisher Scientific, San Jose, CA, USA) equipped with a binary solvent pump, column oven, auto-sampler
and UV detector. A 10-μL aliquot of each sample solution was injected and analyzed on a Sunfire C18
column (150 mm × 4.6 mm, 3.5 μm, Waters, MA, USA). The separation was conducted at 30 °C (column
temperature) using a gradient elution method with 0.5% formic acid in distilled water (solvent A) and
0.1% formic acid in acetonitrile (solvent B). The solvent gradient in volumetric ratios was set as follows:
0–10 min at 88% A; 10–42 min from 88% A to 80% A; 42–55 min from 80% A to 70% A; 55–63 min
from 70% A to 40% A; 63–64 min from 40 % A to 88% A; and 64–65 min at 88% A. The flow rate was
0.6 mL/min and the effluents were monitored at 350 nm.
3.5. Identification of Flavonoids
Flavonoids were identified using a Thermo Accela 600 HPLC system with a UV detector coupled to
a TSQ Quantum Access MAX triple-stage quadropole mass spectrometer (Thermo Fisher Scientific).
Electrospray ionization (ESI) was applied in the negative ion mode (NI) for the MS analysis. The
operation conditions of mass analysis were set as follows: capillary temperature, 350 °C; vaporizer
temperature, 300 °C; sheath gas (N2) pressure, 40 arbitrary units; auxiliary gas (N2) pressure, 10 arbitrary
units; spray voltage, 3 kV. The mass spectra were recorded in the mass range from m/z 150 to 1500. The
Molecules 2015, 20 10561
MS/MS spectra were obtained using the Data-Dependent mode and the collision energy was set as
following: collision energy (CE), 10 V; collision energy grad (CE grad), 0.035 V/m.
3.6. Determination of Antioxidant Activity of Flavonoids
3.6.1. DPPH Free Radical Scavenging Activity
DPPH scavenging activity was determined by the method described by Brand-Williams et al. with
slight modifications [34]. Ten μL of appropriately diluted sample or Trolox solution (31.25–1000 μM)
was added to 190 μL of DPPH solution (final concentration was 0.1 mM in methanol) in a 96-well plate.
Then, the sample mixture was shaken gently and kept in the dark at room temperature for 30 min.
Thereafter, the absorbance at 517 nm was measured and methanol was used for the baseline correction
with a multifunctional microplate reader. The DPPH radical scavenging activity of extracts was
calculated from the standard curve of Trolox and expressed as micromoles of Trolox equivalents (TE)
per gram of sample (μmol TE/g). In addition, to determine the IC50 of samples on DPPH, six different
concentrations were used. Following the same procedure above, the methanol instead of sample was
made as blank controls, while BHT and Trolox were used as positive controls. The ability to scavenge
the DPPH radical was calculated as a percentage according to the following equation:
DPPH-scavenging effect (%) = [(ADPPH AS)/ADPPH] × 100 (1)
where ADPPH = absorbance of control, AS = absorbance of sample), and the IC50 value was determined
to be the effective concentration at which DPPH radicals were inhibited by 50%.
3.6.2. ABTS Free Radical Scavenging Activity
ABTS free radical scavenging activity was determined according to the method adopted by Zou et al.
with some minor modifications [33]. The ABTS assay is based on the capacity to quench ABTS radical
cationic (ABTS+) formation relative to Trolox. Briefly, a solution of ABTS+ was prepared by mixing
equal volumes of potassium persulfate (4.9 mM in H2O) and ABTS (7 mM in H2O), and the solution
was incubated in the dark for 12–16 h. The radical was stable in this form for more than two days when
stored in the dark at room temperature. The ABTS+ solution was then diluted with 80% ethanol to obtain
an absorbance of 0.700 ± 0.005 at 734 nm. Afterwards, ten microliters of appropriately diluted samples
was added to 190 μL of ABTS+ solution in a 96-well plate. The mixture was incubated in the dark at
room temperature for 30 min and then the absorbance was recorded at 734 nm. Trolox was used as
standard, and a standard calibration curve was obtained for Trolox at concentrations ranging from
31.25 μM to 500 μM. The ABTS free radical scavenging activity of samples was calculated from the
standard curve of Trolox and expressed as micromoles of Trolox equivalents (TE) per gram of sample
(μmol·TE/g). The scavenging activities of different concentrations of samples against ABTS+ were also
measured to calculate the IC50, and the procedure was similar to the DPPH scavenging method
described before.
Molecules 2015, 20 10562
3.6.3. Ferric Reducing/Antioxidant Power (FRAP) Assay
FRAP assay was performed as described previously by Benzie and Strain [35]. This method measures
the change in absorbance at 593 nm owing to the formation of a blue colored ferrous 2,4,6-tripyridyl-s-
triazine complex (Fe2+-TPTZ) from colorless oxidized ferric form (Fe3+-TPTZ) by the action of
electron donating antioxidants. The stock solutions included 300 mM acetate buffer, pH 3.6 (3.1 g
C2H3NaO2·3H2O and 16 mL C2H4O2), 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl3·6H2O
solution. The working solution was prepared by mixing 10 volumes of acetate buffer, 1 volume of TPTZ
solution, and 1 volume of FeCl3·6H2O solution and then warmed at 37 °C before use. Then, 10 μL of
properly diluted samples and 30 μL of distilled water was added to 260 μL of freshly prepared FRAP
reagent in a 96-well plate and mixed thoroughly. The mixture was incubated at 37 °C for 10 min, and
the absorbance was measured at 593 nm. The FRAP value was calculated and expressed as millimoles
of Fe2+ equivalents per 100g of sample (mmol Fe2+equiv/100 g) based on a calibration curve plotted
using FeSO4·7H2O as standard at a concentration ranging from 0.125 to 2 mM. All solutions were freshly
made and used on the day of preparation.
3.7. Statistical Analysis
Data were expressed as the mean ± standard deviation of triplicate measurements. The data were
statistically analyzed using statistical software, OriginPro 8.6.
4. Conclusions
To extend our research on the correlations and underlying mechanisms between chemical components
and their bioactivities, a more efficient macroporous resin chromatography with much better resolution
and compatibility coupled with HPLC-MS was developed for the simultaneous separation and biochemical
analysis of flavonoids from the complex extracts of lotus leaves. Altogether, fourteen flavonoids from
lotus leaves were identified in this work, which greatly improved the previous LC-MS/MS method. More
importantly, the newly developed method has led to some significant new findings. Among fourteen
flavonoids identified, quercetin 3-O-rhamnopyranosyl-(12)-gluco- pyranoside, quercetin 3-O-arabinoside,
diosmetin 7-O-hexose and isorhamnetin 3-O-arabino- pyranosyl-(12)-glucopyranoside were identified
in lotus leaves for the first time. In addition, this macroporous resin chromatography method is very
compatible with flexible downstream biological activity studies, and greatly facilitated the antioxidant
activity screening in this study. It is expected that with some slight modifications this new method will
prove useful to explore more important applications in food and pharmaceutical industries.
This work was jointly supported by “the Hundred Talents Program” from Chinese Academy of
Sciences (Grant No. 29Y429291a0129 to M. Guo), The Knowledge Innovation Project of Chinese
Academy of Sciences (Grant No. Y455421Z02), and the Key Laboratory of Plant Germplasm
Enhancement and Specialty Agriculture of Wuhan Botanical Garden, Chinese Academy of Sciences.
Molecules 2015, 20 10563
Author Contributions
Ming-Quan Guo and Ming-Zhi Zhu conceived and designed the experiments; Ming-Zhi Zhu,
Wei Wu, Li-Li Jiao and Ping-Fang Yang performed the experiments or helped with the data analysis;
Ming-Zhi Zhu and Ming-Quan Guo wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds (quercetin 3-O-galactoside (hyperoside), quercetin
3-O-glucoside (isoquercitrin), kaempferol 3-O-glucoside (astragalin)) are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
... Strong absorbance peaks were obtained for each of the three compounds. The respective peaks within the NNL ethanolic extract eluted at 41.3, 43.8, and 50.5 min (Figure 1a [18,19]. The peaks of the standard were eluted at 41.3, 44.1, and 50.4 min, respectively, confirming that Quercetin 3-O-galactoside (hyperoside), Quercetin 3-O-β-D-glucuronide (miquelianin), and Quercetin 3-O-glucoside (isoquercetin) were the main components of the NNL ethanolic extract that we used for the experiments (Figure 1b,c). ...
... Comparative HPLC chromatograms of NNL ethanolic extract were carried out. Absorbance was measured at 350 nm based on previously obtained information [19]. Strong absorbance peaks were obtained for each of the three compounds. ...
... previously known standard components of NNL extract[18,19]. The peaks of the standard were eluted at 41.3, 44.1, and 50.4 min, respectively, confirming that Quercetin 3−O−galactoside (hyperoside), Quercetin 3−O−β−D−glucuronide (miquelianin), and Quercetin 3−O−glucoside (isoquercetin) were the main components of the NNL ethanolic extract that we used for the experiments(Figure 1b,c). ...
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Muscle atrophy is characterized by a decline in muscle mass and function. Excessive glucocorticoids in the body due to aging or drug treatment can promote muscle wasting. In this study, we investigated the preventive effect of Nelumbo nucifera leaf (NNL) ethanolic extract on muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, in mice and its underlying mechanisms. The administration of NNL extract increased weight, cross-sectional area, and grip strength of quadriceps (QD) and gastrocnemius (GA) muscles in DEX-induced muscle atrophy in mice. The NNL extract administration decreased the expression of muscle atrophic factors, such as muscle RING-finger protein-1 and atrogin-1, and autophagy factors, such as Beclin-1, microtubule-associated protein 1A/1B-light chain 3 (LC3-I/II), and sequestosome 1 (p62/SQSTM1) in DEX-injected mice. DEX injection increased the protein expression levels of NOD-like receptor pyrin domain-containing protein 3 (NLRP3), cleaved-caspase-1, interleukin-1beta (IL-1β), and cleaved-gasdermin D (GSDMD), which were significantly reduced by NNL extract administration (500 mg/kg/day). In vitro studies using C2C12 myotubes also revealed that NNL extract treatment inhibited the DEX-induced increase in autophagy factors, pyroptosis-related factors, and NF-κB. Overall, the NNL extract prevented DEX-induced muscle atrophy by downregulating the ubiquitin–proteasome system, autophagy pathway, and GSDMD-mediated pyroptosis pathway, which are involved in muscle degradation.
... Nelumbo nucifera (Sacred lotus) is an aquatic species belonging to Nelumbonaceae having herbal remedial benefits in every part of the plant (44). Widely used in Ayurveda and traditional medicines (22, 23, 45), the lotus is native to tropical and subtropical zones of Asia and almost all parts of it, such as flower, seed, leaf, stem, and root are edible (44) whose extract contains various phytochemicals, including alkaloids, flavonoids, phenolic acids, and steroids (22, 23, 45-49), which promote antioxidant (23,45,50) anti-inflammatory (51,52), anti-diabetic (24), anti-obesity (25), and anti-cancer (26) activities. These biological activities are beneficial for individuals with declined immune functions, especially to the aged and advanced age people (53,54), and immunosuppression in diabetic patients (27). ...
... The measured Fe 3+ reduction and FRAP values reached 0.73 mg RE /mL and 0.71 mmol/L Fe 2+ (Figure 5b), while the FRAP value of 0.1 mg/mL rutin was 0.74 mmol/L Fe 2+ , which could be referred to as a reference. As some reports have shown, the ethanol extract of lotus leaves exhibited antioxidant activities in DPPH scavenging activity, ABTS scavenging activity, and the FRAP assay [51]. Compared to water extraction, methanol extraction had higher antioxidant activities [52]. ...
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The discovery of a green extraction solvent for natural plants could promote related research. In this study, deep eutectic solvents (DES) were used as green solvents coupled with an ultrasound-assisted extraction method (UAE) to extract flavonoids from lotus leaves. Thirty-four different DES were performed and choline chloride/urea with 40% water was chosen as the most promising one, and the related parameters in the procedures were optimized, resulting in the highest extraction amount of flavonoids in lotus leaves. D-101 was selected from four macroporous resins to separate the flavonoids from DES. Moreover, DES could be recycled and efficiently reused four times with satisfactory performances. In addition, the lotus leaf flavonoids from the DES extract exhibited antioxidant activities in five kinds of assays including DPPH, ABTS, Fe3+ reducing, FRAP, and Fe2+ chelating. It also showed antibacterial activities on Staphylococcus aureus and Escherichia coli bacterial strains with minimal inhibitory concentrations at 1666 μg/mL and 208 μg/mL, respectively. In the HPLC analysis, the three main components in the DES extract were identified as astragalin, hyperoside, and isoquercitrin. In conclusion, the developed UAE-DES followed by macroporous resin treatment could become an efficient and environmentally friendly extraction and enrichment method for flavonoids from lotus leaves and other natural products.
... Likewise, compounds 74, 92, 124, and 148 were deduced to be quercetin derivatives; compound 78 was quercetin 3rutinoside 7-rhamnoside [14]; and compounds 100 and 108 were quercetin 3-O-(6″-galloyl)-β-D-glucopyranoside isomers. Compound 105 was characterized as quercitrin 3-O-glucuronide, and compounds 116 and 122 were quercitrin 3-O-arabinoside isomers [15][16][17]. ...
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Diospyros lotus L, F. Ebenaceae, is an edible fruit that is widely distributed in China and other Asian countries. Presently, Diospyros lotus L can be used to treat patients with diabetes; however, its chemical composition and pharmacological profiles remain to be elucidated. This study investigated the potential bioactive compounds of Diospyros lotus L and their mechanisms of action using LC-MS and network pharmacology analysis. First, the components of Diospyros lotus L were identify using a reliable strategy for UHPLC-Q-Exactive Orbitrap mass spectrometry combined with parallel reaction monitoring (PRM) in the negative ion mode. Second, a network pharmacology study, including target gene prediction and functional enrichment, was applied to screen the main quality markers of Diospyros lotus L and explore its potential mechanism for the treatment of diabetes. The results showed that a total of 159 compounds were identified from Diospyros lotus L, among which, 140 were reported for the first time. Furthermore, 40 active components, such as quercetin, luteolin, and kaempferol, were proposed as active components of Diospyros lotus L for the treatment of diabetes based on network pharmacology analysis. In addition, 92 relevant antidiabetic targets were mainly related to positive regulation of transcription from the RNA polymerase II promoter, extracellular space, and protein binding, suggesting the involvement of TNF, PI3K-Akt, and HIF-1 signaling pathways in the antidiabetic effect of Diospyros lotus L. Our results may provide a useful approach to identify potential active components and molecular mechanisms of Diospyros lotus L for the treatment of diabetes.
... Metode penentuan flavonoid total mengacu pada metode yang dikembangkan Zhu et al. (2015), dengan beberapa modifikasi. Sampel sebanyak 10 µL dimasukkan ke dalam sumuran plat mikro, lalu ditambahkan etanol 60 µL dan 10 µL AlCl 3 (10% b/v), serta 10 µL kalium asetat (1 M), selanjutnya ditambahkan aqua destillata 120 µL. ...
Diabetes mellitus (DM) is a disease caused by lacking of insulin production or by the inability of cells to respond to insulin (insulin resistance). According to the International Diabetes Federation, diabetes cases in the world reach 425 millions and are predicted to increase to 625 millions by 2045. The trend of increasing cases and death rates due to diabetes needs a special attention, especially in the pattern of it’s treatment. Diabetes treatment using natural ingredients is one of the most researched fields in the world because it is effective and safe. Curculigo latifolia Dryand. ex W.T. Aiton and Curculigo orchioides Gaertn. belonging to the family Hypoxidaceae, annual herbs with lanceolate-shaped leaves or parallel lanceolate arranged in a rosette, with yellow flowers, very short stems, and have a long cylindrical rhizome. A total of 39 species of this genus are accepted in the World Checklist of Selected Plant Families (WCSP 2020), including these two species. Both species are known as traditional medicinal plants in various tropical regions. Rhizome of Curculigo spp. is one of the raw material sources for traditional medicine to treat DM; this pharmacological effect comes from secondary metabolites. Those compounds are distributed and accumulated in certain secretory structures within the plant. However, the activities of the active compounds in such diverse plant organs are very difficult to be determined in a short time, as well as its pharmacokinetic and pharmacodynamics parameters. In addition, compounds produced under normal conditions in the nature are very low. Therefore, this study aimed to determine the distribution of secretory structures and the producing and/or accumulating sites of the bioactive compounds through histochemical tests, to determine which bioactive compounds contribute the most to diabetes mellitus, especially in antioxidant activity and α-glucosidase inhibition, and to determine their pharmacokinetics and pharmacodynamics parameters. In addition, this research was also carried out to produce callus and micropropagate the plants, as well as to ensure the existence of those bioactive compounds in in vitro cultured callus. Determination of secretory structure using cross sections of fresh samples according to plant anatomical procedures and histochemical analysis using several reagents were performed to detect groups of metabolites. Determination of bioactive compounds was done using an analysis combination on biological activities (antioxidants and α-glucosidase inhibition) with metabolite fingerprint using FTIR and metabolite profiling with UHPLC-Q-Orbitrap HRMS-based metabolomic and chemometric techniques using partial least squares regression analysis (PLSR). Pharmacokinetics and pharmacodynamics parameters were determined using Lipinski's rule of five, pharmacological networks using Cytoscape, and molecular docking with PyRx, PyMOL, and BIOVIA Discovery Studio. Callus production and micropropagation began with explant sterilization using environmental-friendly sterilants. Callus initiation and organogenesis were induced by various concentrations of auxins and cytokinin. Metabolomic analysis based on metabolite profiling using UHPLC-Q-Orbitrap HRMS and chemometric techniques using principal component analysis (PCA) were carried out to identify the compounds in the callus and plantlet’s leaves. The anatomical and histochemical analysis of fresh tissues showed that all organs contained secretory structures that accumulated various metabolites. The secretory structures identified in the roots, rhizomes, petiole, and leaves of these two species were secretory cavities and idioblasts. The group of compounds identified were phenols, alkaloids, terpenes, essential oils, and lipophilic. They were also spread over some common tissues of the organs. Based on metabolomic and chemometric analysis the main compounds contributing in antioxidant and α-glucosidase inhibition activities were notified from the phenol group, such as curculigoside B, orchioside B; 2,4-Dichloro-5-methoxy-3-methylphenol, orcinol glucoside; 1,1-Bis-(3,4-dihydroxyphenyl)-1-(2-furan)-methane; from the terpene group, such as: curculigosaponin G, H, and I; from the norlignan group, (1S,2R)-O-Methylnyacoside; and from the aldehyde group, 5-hydroxymethylfural, while the functional groups included O–H, C=O, C–O, C–H. These compounds were accumulated more abundantly in the leaves of C. latifolia (DLSP) from Sinjai-Palangka and C. orchioides (DOGM) from Gowa-Malakaji. Pharmacokinetic parameters showed that 33 out of the 79 compounds were able to be absorbed properly, while some compounds did not meet the requirements. The latter compounds must be converted into aglycones if they will be used as medicinal substances. The cynanuriculoside ligand A_qt based on pharmacological network analysis and molecular docking was able to interact pharmacodynamically with hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) target via 6NJ7 receptor, resulting an affinity of –12.0 (kcal mol–1), with amino acid residues in the form of Ala 226, Leu 126, Val 180, Tyr 183, Leu 215, Ser 170, Ile 121, and Val 168. The sterilization of explants with the lowest concentrations of sterilizing agents and a short contact time with the explants produced 90% sterile cultures. The best combination of plant growth regulators (PGRs) for callus induction in C. latifolia and C. orchioides were BAP : IBA at 3 : 5 and 5 : 3 mg L–1, respectively. The callus were green and white, with a compact consistency. Those combinations of PGRs also regenerated shoots and roots in both species. The secretory structures found in the callus were secretory cavities and idioblast cells. In the callus of C. latifolia, phenol was identified in the organogenic parts and epithelium cells of the secretory cavities, and the essential oils were in idioblast cells; while C. orchioides’ callus contained phenol in the organogenic parts only. The compounds that had contribution in antioxidant and α-glucosidase inhibition activities, such as 1,1-Bis-(3,4-dihydroxyphenyl)-1-(2-furan)-methane, (1S,2R)-O-Methylnyacoside; 2,4-Dichloro-5-methoxy-3-methylphenol, curculigoside B, curculigosaponin G, H, and I; orchioside B, and orcinol glucoside were also identified in the callus and plantlet’s leaves. Most of them belong to the phenol group. The general conclusion of this study is that histochemical techniques revealed that there were differences in the accumulation sites of compounds among organs of Curculigo spp. Histochemically, phenolic compounds were identified in the rhizome, petiole, and leaves of C. latifolia, while in C. orchioides they were only identified in the rhizome. Phenolics were also found in the organogenic callus of these two species. From the metabolomic-chemometric analysis, compounds that contributed greatly to the antioxidant and α-glucosidase inhibition activities were accumulated in the leaves of both species. From the pharmacological network and molecular docking approaches, cynanuriculoside A_qt, curculigosaponin L_qt, and curculigenin B were confirmed to have potential for the treatment of diabetes mellitus. The compounds found in the plant’s organs of C. latifolia and C. orchioides that contribute greatly in antioxidant and α-glucosidase inhibition activities were also identified in the callus and plantlet’s leaves resulted from in vitro cultures. Some of which even demonstrated higher concentration (peak area) than those of the original plant organs.
... Nelumbo nucifera (Sacred lotus) is an aquatic species belonging to Nelumbonaceae having herbal remedial benefits in every part of the plant (44). Widely used in Ayurveda and traditional medicines (22, 23, 45), the lotus is native to tropical and subtropical zones of Asia and almost all parts of it, such as flower, seed, leaf, stem, and root are edible (44) whose extract contains various phytochemicals, including alkaloids, flavonoids, phenolic acids, and steroids (22, 23, 45-49), which promote antioxidant (23,45,50) anti-inflammatory (51,52), anti-diabetic (24), anti-obesity (25), and anti-cancer (26) activities. These biological activities are beneficial for individuals with declined immune functions, especially to the aged and advanced age people (53,54), and immunosuppression in diabetic patients (27). ...
Full-text available
Tea is an important beverage consumed worldwide. Of the different types of tea available, herbal tea is an important beverage consumed owing to its popularity as a drink and stress relieving factors, several different herbal concoctions made from seeds, leaves, or roots are currently consumed and sold as herbal teas. The herbal teas are not the usual tea but “tisanes.” They are caffeine free and popular for their medicinal property or immune boosters. Herbal tea formulations are popularly sold and consumed by millions owing to their health benefits as they are rich in antioxidants and minerals. However, plants are also known to contain toxic and anti-nutritional factors. Anti-nutritional factors are known to interfere with the metabolic process and hamper the absorption of important nutrients in the body. These anti-nutritional factors include saponins, tannins, alkaloids, oxalates, lectins, goitrogens, cyanogens, and lethogens. These chemicals are known to have deleterious effects on human health. Therefore, it is important to understand and assess the merits and demerits before consumption. Also, several techniques are currently used to process and reduce the anti-nutrients in foods. This review is focused on comparing the contents of various anti-nutritional factors in some underutilized plants of North-East India used as herbal tea along with processing methods that can be used to reduce the level of these anti-nutrients.
... In many Asian countries including Thailand, Japan, China, India, and Sri Lanka, sacred lotus (Nelumbo nucifera Gaertn., Nelumbonaceae) is used for popular herbal teas and traditional medicines [12,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. This aquatic medicinal plant has a long history of use in foods and traditional medicines, in particular in the form of herbal teas [18,20,25,[34][35][36][37]. ...
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Stamen tea from Nelumbo nucifera Gaertn. (or the so-called sacred lotus) is widely consumed, and its flavonoids provide various human health benefits. The method used for tea preparation for consumption, namely the infusion time, may affect the levels of extractable flavonoids, ultimately affecting their biological effects. To date, there is no report on this critical information. Thus, this study aims to determine the kinetics of solid liquid extraction of flavonoid from sacred lotus stamen using the traditional method of preparing sacred lotus stamen tea. Phytochemical composition was also analyzed using high-performance liquid chromatography (HPLC). The antioxidant potential of stamen tea was also determined. The results indicated that the infusion time critically affects the concentrations of flavonoids and the antioxidant capacity of sacred lotus stamen tea, with a minimum infusion time of 5–12 min being required to release the different flavonoids from the tea. The extraction was modeled using second order kinetics. The rate of release was investigated by the glycosylation pattern, with flavonoid diglycosides, e.g., rutin and Kae-3-Rob, being released faster than flavonoid monoglycosides. The antioxidant activity was also highly correlated with flavonoid levels during infusion. Taken together, data obtained here underline that, among others, the infusion time should be considered for the experimental design of future epidemiological studies and/or clinical trials to reach the highest health benefits.
Nelumbo nucifera Gaertn, commonly known as lotus, is a genus comprising perennial and rhizomatous aquatic plants, found throughout Asia and Australia. This review aimed to cover the biosynthesis of flavonoids, alkaloids, and lipids in plants and their types in different parts of lotus. This review also examined the physiological functions of bioactive compounds in lotus and the extracts from different organs of the lotus plant. The structures and identities of flavonoids, alkaloids, and lipids in different parts of lotus as well as their biosynthesis were illustrated and updated. In the traditional medicine systems and previous scientific studies, bioactive compounds and the extracts of lotus have been applied for treating inflammation, cancer, liver disease, Alzheimer's disease, etc. We suggest future studies to be focused on standardization of the extract of lotus, and their pharmacological mechanisms as drugs or functional foods. This review is important for the lotus-based food processing and application.
Lotus (Nelumbo nucifera Gaertn.) is an aquatic perennial crop planted worldwide and its leaf (also called “He-Ye”) has therapeutic effects on obesity. However, whether the underlying mechanism leads to increased energy expenditure by activation of brown adipocytes has not been clarified. Here, murine C3H10T1/2 mesenchymal stem cells (MSCs) were employed to investigate the effects of ethanol extracts from lotus leaf (LLE) on brown adipocytes formation and the underlying molecular mechanisms. The results showed LLE was rich in polyphenols (383.7 mg/g) and flavonoids (178.3 mg/g), with quercetin 3-O-glucuronide (Q3G) the most abundant (128.2 μg/mg). In LLE-treated C3H10T1/2 MSCs, the expressions of lipolytic factors (e.g., ATGL, HSL, and ABHD5) and brown regulators (e.g., Sirt1, PGC-1α, Cidea, and UCP1) were significantly upregulated compared to that in the untreated MSCs. Furthermore, LLE promoted mitochondrial biogenesis and fatty acid β-oxidation, as evidenced by increases in the expression of Tfam, Cox7A, CoxIV, Cox2, Pparα, and Adrb3. Likewise, enhanced browning and mitochondrial biogenesis were also observed in Q3G-stimulated cells. Importantly, LLE and Q3G induced phosphorylation of AMPK accompanied by a remarkable increase in the brown fat marker UCP1, while pretreatment with Compound C (an AMPK inhibitor) reversed these changes. Moreover, stimulating LLE or Q3G-treated cells with CL316243 (a beta3-AR agonist) increased p-AMPKα/AMPKα ratio and UCP1 protein expression, indicating β3-AR/AMPK signaling may involve in this process. Collectively, these observations suggested that LLE, especially the component Q3G, stimulates thermogenesis by activating brown adipocytes, which may involve the β3-AR/AMPK signaling pathway.
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The Asteraceae is a large family, rich in ornamental, economical, and medicinally valuable plants. The current study involves the analytical and pharmacological assessment of the methanolic extracts of three less investigated Asteraceae plants, namely Echinops ritro, Centaurea deflexa, and Tripleurospermum decipiens, obtained by three different extraction methodologies viz. maceration (MAC), ultrasound-assisted extraction (UAE), and homogenizer-assisted extraction (HAE). LC-MS- MS analysis of E. ritro, C. deflexa, and T. decipiens extracts led to the identification of ca. 29, 20, and 33 metabolites, respectively, belonging to flavonoids, phenolic acids, and fatty acids/amides. Although there were significant differences in the quantitative metabolite profiles in the extracts of E. ritro and T. decipiens based on the used extraction method, no significant variation was observed in the extracts of C. deflexa in the three implemented extraction techniques. The antioxidant activities of the nine extracts were assessed in vitro using six different assays viz. DPPH, ABTS, CUPRAC, FRAP, PDA, and metal chelation assay (MCA). The HAE/UAE extracts of E. ritro and the UAE/ MAC extracts of C. deflexa displayed the highest antioxidant activity in the DPPH assay, while the UAE extract of T. decipiens showed the strongest antioxidant activity in both the CUPRAC and MCA assays. The enzyme inhibitory activities of the nine extracts were studied in vitro on five different enzymes viz. tyrosinase, α-amylase, α-glucosidase, acetylcholinesterase (AChE), and butyrylcholinestrase (BChE), affecting various pathological diseases. Concerning C. deflexa, its MAC /UAE extracts showed the strongest inhibition on α-amylase, while its UAE/HAE extracts displayed strong inhibitory power on AChE. However, no significant difference was observed on their effects on tyrosinase or BChE. For T. decipiens, its UAE/HAE showed potent inhibition to α-glucosidase, MAC/ HAE significantly inhibited AChE and BChE, while UAE could strongly inhibit tyrosinase enzyme. For E. ritro, all extracts equally inhibited α-amylase and α-glucosidase, MAC/HAE strongly affected tyrosinase, HAE/MAC best inhibited BChE, while HAE inhibited AChE to a greater extent. Chemometric analysis using PCA plot was able to discriminate between the plant samples and between the implemented extraction modes. The in vitro enzyme inhibitory activities of the extracts were supported by in silico data, where metabolites, such as the lignan arctiin and the flavonoid vicenin-2, dominating the extract of C. deflexa, displayed strong binding to AChE. Similarly, chlorogenic and dicaffeoyl quinic acids, which are some of the major metabolites in the extracts of E. ritro and T. decipiens, bound with high affinity to α-glucosidase
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Flavonoids are rich in Juglans regia L. leaves. They have potent antioxidant properties, which have been related to regulating immune function and enhancing anticancer activity. Herein, qualitative and quantitative determination of flavonoids from J. regia leaves was carried out using high performance liquid chromatography coupled with tandem mass spectrometry with electrospray ionization and negative ion detection (HPLC-ESI-MS/MS) by comparison of the retention times and mass spectral fragments with standard substances or related literatures. Seventeen compounds were identified and major components are quercetin-3-O-rhamnoside (453.11 íµí¼‡g/g, dry weight), quercetin-3-O-arabinoside (73.91 íµí¼‡g/g), quercetin-3-O-xyloside (70.04 íµí¼‡g/g), kaempferol-O-pentoside derivative (49.04 íµí¼‡g/g), quercetin-3-O-galactoside (48.61 íµí¼‡g/g), and kaempferol-O-pentoside (48.46 íµí¼‡g/g). The in vitro intracellular antioxidation indicated that flavonoids from J. regia leaves could reduce the reactive oxygen species (ROS) level in RAW264.7 cells and showed good radical scavenging activities. These results proved to be more related to the flavonoids that could be considered in the design of new formulations of dietary supplements or functional foods.
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ABSTRACT Aging and neurodegenerative diseases share oxidative stress cell damage and depletion of endogenous antioxidants as mechanisms of injury, phenomenons that occurring at different rate in each process. Nevertheless, as the central nervous system (CNS) consists largely of lipids and has a poor activity of catalase, a low amount of superoxide dismutase and is rich in iron, its cellular components are damaged easily by over production of free radicals in any of these physiologic or pathologic conditions. Thus, antioxidants are needed to prevent the formation and oppose the free radicals damage to DNA, lipids, proteins, and other biomolecules. Due to endogenous antioxidant defenses are inadequate to prevent damage completely, different efforts have been undertaken in order to increase the use of natural antioxidants and to develop antioxidants that might ameliorate neural injury by oxidative stress. In this context, natural antioxidants like flavonoids (quercetin, curcumin, luteolin and catechins) and magnolol and honokiol, are showing to be efficient inhibitors of the oxidative process and seem to be a better therapeutic option than the traditionally ones (vitamin C, E and β-carotene) in various models of aging and injury in vitro and in vivo conditions. Thus, the goal of the present review is to discuss the molecular basis, mechanisms of action, functions, and targets of flavonoids, magnolol, honokiol and traditional antioxidants with the aim to obtained better results when they are prescribing on aging and neurodegenerative diseases.
Flavones have antioxidant, anti-proliferative, anti-tumor, anti-microbial, estrogenic, acetyl cholinesterase, anti-inflammatory activities and are also used in cancer, cardiovascular disease, neurodegenerative disorders etc. Due to the wide range of biological activities of flavones have generated interest among medicinal chemists. This review may provide an opportunity to scientists of medicinal chemistry discipline to design selective, optimize as well as poly-functional flavone derivatives for the treatment of multi-factorial diseases.
Enrichment and purification of total flavonoids from Flos Populi extracts were studied using five macroporous resins. The static tests indicated that NKA-9 resin was appropriate and its adsorption data were well fitted to the Langmuir and Freundlich isotherms. To optimize the separation process, dynamic adsorption and desorption tests were carried out. The optimal adsorption parameters were initial concentrations in sample solution of 7.64mg/mL, pH of 5.0, sample loading amount of 2.3BV, flow rate of 2BV/h, temperature of 25°C. The optimal desorption parameters were deionized water and 20% ethanol each 5BV, then 60% ethanol of 10 BV, flow rate of 2BV/h. After one run treatment with NKA-9 resin, the content of total flavonoids in the product increased from 11.38% to 53.41%, and the recovery yield was 82.24%. The results showed that NKA-9 resin revealed a good ability to enrichment total flavonoids from Flos Populi, and the method can be referenced for the enrichment of total flavonoids from other materials. The antioxidant activities of the purified flavonoids were further evaluated in vitro. It showed that the DPPH radical scavenging increased from 59.46% to 82.63% at different concentrations (0.06-0.14mg/mL). At different concentrations (0.6-1.4mg/mL), the hydroxyl radical scavenging increased from 35.39% to 74.12%. Moreover, the reducing ability and total oxidant capacity appeared to be dose-dependent of flavonoids. It indicated that the purified flavonoids can be used as a source of potential antioxidant.
A novel method has been developed for analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by using high-performance liquid chromatography (HPLC)–photodiode array detection (DAD)–electrospray mass spectrometry (ESI-MS). The method was carried out by using a Shimadzu VP-ODS column with a gradient solvent system of 0.1% triethylamine aqueous solution–acetonitrile. N-Nornuciferine, O-nornuciferine, nuciferine, and roemerine were identified with authentic standard compounds and with MS-spectra. The contents of these compounds were measured by employing DAD. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 30–90pg.
Oxidative stress is associated with several pathologies like cardiovascular, neurodegenerative, cancer and even aging. It has been suggested that a diet rich in antioxidants would be beneficial to human health and a lot of interest is focused on the determination of antioxidant capacity of natural products. Different chemical methods have been developed including the popular ORAC that evaluates the potential of a sample as inhibitor of a target molecule oxidation. Chemical-based methods are useful for screening, they are low cost, high-throughput and yield an index value (expressed as equivalents of Trolox) that allows comparing and ordering different products. More recently, nanoparticles-based assays have been developed to sense the antioxidant power of natural products. However, the antioxidant capacity indexes obtained by chemical assays cannot extrapolate the performance of the sample in vivo. Considering that antioxidant action is not limited to scavenging free radicals but includes upregulation of antioxidant and detoxifying enzymes, modulation of redox cell signaling and gene expression, it is necessary to move to cellular assays in order to evaluate the potential antioxidant activity of a compound or extract. Animal models and human studies are more appropriate but also more expensive and time-consuming, making the cell culture assays very attractive as intermediate testing methods. Cellular antioxidant activity (CAA) assays, activation of redox transcription factors, inhibition of oxidases or activation of antioxidant enzymes are reviewed and compared with the classical in vitro chemical-based assays for evaluation of antioxidant capacity of natural products.