Polyphenolic compounds from Malus hupehensis and their free radical scavenging effects

Article abstract of DOI:10.1080/14786419.2017.1367784

One new 4-chromanone glycoside, 5-O-β-d-glucopyranoside-4-chromanone (1), together with 21 known polyphenols, was isolated from the leaves of Malus hupehensis. Their structures were elucidated on the basis of extensive spectroscopic methods including NMR

Full text of DOI:10.1080/14786419.2017.1367784

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Polyphenolic compounds from Malus hupehensis
and their free radical scavenging effects
Qingwen Hu, Ying-Ying Chen, Qi-Yang Jiao, Afsar Khan, Jimiao Shan, Gui-
Dong Cao, Feng Li, Chao Zhang & Hong-Xiang Lou
To cite this article: Qingwen Hu, Ying-Ying Chen, Qi-Yang Jiao, Afsar Khan, Jimiao Shan,
Gui-Dong Cao, Feng Li, Chao Zhang & Hong-Xiang Lou (2017): Polyphenolic compounds from
Malus hupehensis and their free radical scavenging effects, Natural Product Research, DOI:
10.1080/14786419.2017.1367784
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Date: 14 September 2017, At: 05:01

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1367784
Polyphenolic compounds from Malus hupehensis and their
free radical scavenging effects
Qingwen Hua, Ying-Ying Chena,b, Qi-Yang Jiaob, Afsar Khanc, Jimiao Shanb,
Gui-Dong Caob, Feng Lib, Chao Zhangb and Hong-Xiang Loua
aKey laboratory of chemical Biology of Ministry of education, department of Natural Products chemistry,
school of Pharmaceutical sciences, shandong university, Jinan, People’s republic of china; bNatural Products
laboratory, research and development department, reyoung Pharmaceutical co., ltd., Jinan, People’s
c
republic of china; department of chemistry, coMsats Institute of Information technology, abbottabad,
Pakistan
ABSTRACT
ARTICLE HISTORY
received 7 June 2017
accepted 31 July 2017
One new 4-chromanone glycoside, 5-O-β-d-glucopyranoside-4-
chromanone (1), together with 21 known polyphenols, was isolated
from the leaves of Malus hupehensis. Their structures were elucidated
on the basis of extensive spectroscopic methods including NMR (1D
and 2D), mass (ESIMS and HRESIMS), IR, and by comparison with the
data reported in the literature. Some of the isolated compounds were
screened for antioxidant activity. Compounds 18 and 14 exhibited
significant antioxidant activities with SC50 values 2.73 and 2.91 μg/mL,
respectively, while 17, 19, 11, 7, 20, 22, 12 and 13 exhibited moderate
activities with SC50 values ranging from 5.24–11.86 μg/mL. The HPLC
fingerprint profiles of the leaves and fruits extracts were also analysed,
which showed that the constituents were almost the same in both
the extracts except for the content of phlorizin which was present in
higher amount in the leaves.
KEYWORDS
Malus hupehensis;
4-chromanone; polyphenols;
antioxidant activity; free
radical scavenging
1. Introduction
Previous phytochemical investigation on the genus Malus has revealed the secondary metab-
olites, mainly the polyphenols, including chromones, chalcones, flavones (Williams 1979;
Roemmelt et al. 2003; Seeram et al. 2003; Sultana and Anwar 2008; Mari et al. 2010; Wang
et al. 2013). Researches show that this genus possesses potential pharmacological activities
CONTACT hong-Xiang lou
louhongxiang@sdu.edu.cn
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1367784.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
Q. HU ET AL.
Figure 1. structures of compounds 1–22 from M. hupehensis.
Table 1. dPPh radical scavenging activities of the compounds isolated from M. hupehensis.
Compound
SC50 (μg/mL)
Compound
SC50 (μg/mL)
1
>128
>128
Nta
12
13
14
15
16
17
18
19
20
21
22
10.37
11.86
2.91
21.04
16.29
5.24
2.73
5.88
6.34
Nta
2
3
4
Nta
5
>128
47.70
6.28
>128
>128
>128
5.96
6
7
8
9
10
11
9.07
Edaravone
4.72
aNt: Not tested.
such as antioxidant, antihyperglycaemic, hepatoprotective and antiproliferative activities
(Barbosa et al. 2010; Yang et al. 2010; Xue et al. 2011; Wang et al. 2013). Therefore, this genus
has attracted much attention due to diverse structures of secondary metabolites and signif
-
icant biological activities. Malus hupehensis (Rosaceae) is a well-known medicinal herb
mainly distributed throughout southern China (Delectis Florae Reipublicae Popularis Sinicae
Agenda Academiae Sinicae Edita 1974). The leaves of M. hupehensis have been used as an
important class of tea with a local name ‘‘San-Pi-Guan’’ in Hubei province of China; three
pieces of leaves make one pot of tea. Local people drink‘‘San-Pi-Guan’’for the treatment of
hyperglycaemia (Williams 1979; Shen et al. 2013; Liu et al. 2015). Besides, phlorizin, a main
component of the leaves, is effective to treat diabetes (Rossetti et al. 1987, 1987; Ehrenkranz

NATURAL PRODUCT RESEARCH
3
et al. 2005). Its fruits are sold in the market as health-caring liquor or immersed into the spirit
to make the folk herbal liquor. Due to these uses, we were interested to find out whether
the constituents are different in leaves and fruits. Therefore, we analysed the HPLC fingerprint
profiles of the leaves and fruits extracts. The results showed that the constituents were almost
the same in both the extracts except for the content of phlorizin which was present in higher
amount in the leaves (see Supplementary material). Intrigued by the diverse chemical com-
ponents and potential biological activities, we performed the photochemical investigation
of the leaves of M. hupehensis. As a result, one new 4-chromanone glycoside, together with
21 known compounds, was isolated. Antioxidant activity assay showed that compounds 18
and 14 exhibited significant antioxidative activities with SC50 values 2.73 and 2.91 μg/mL,
respectively, with edaravone as a positive control, while 17, 19, 11, 7, 20, 22, 12 and 13
showed moderate activities with SC50 values ranging from 5.24 to 11.86 μg/mL. The HPLC
fingerprint analysis of the fruits of M. hupehensis showed that they contained mostly
the same active components as those extracted from the leaves (see Supplementary
material).
2. Results and discussion
The molecular formula of compound 1 was assigned as C15H18O9 from the (+)-HRESIMS peak
at m/z 365.0849 [M + Na]+ (Calcd 365.0843), with 7 degrees of unsaturation, which was also
consistent with its 13C and DEPT NMR data. The 13C NMR spectrum resolved 15 carbon res-
onances, of which 6 belonged to a sugar moiety (δC 103.8, 77.6, 75.7, 73.5, 69.7, 60.7) and
the remaining 9 ascribed to a 4-chromanone moiety. The resonances assigned to the 4-chro-
manone unit included two methylenes (δC 65.9, 38.3), two aromatic methines (δC 101.2, 98.5)
and five quaternary carbons (four aromatic δC170.2, 164.2, 160.9, 103.6 and one carbonyl δC
189.0). The structure of the 4-chromanone was established by HSQC and HMBC spectra (see
Table S1 and Figure S1). In addition, the correlations of δH 4.57 (1H, d, J = 7.2 Hz, H-1׳) with
δC 160.9 (C-5) suggested the sugar moiety was linked to the C-5 of aglycone. Acid hydrolysis
of 1 afforded d-glucose on the basis of GC analysis. The β-configuration of d-glucose was
determined by the J coupling constant value (7.2 Hz) of H-1׳. Thus, the structure of 1 was
elucidated as 5-O-β-d-glucopyranoside-4-chromanone.
Besides, 21 known compounds were also isolated and identified as 5,7-dihydroxychro-
mone (2) (Behery et al. 2013), protocatechuate (3) (Zhang et al. 1998), 4-hydroxybenzoic
acid (4) (Saito et al. 1988), salicylamide (5) (Woods and Sherry 2003), phloretin (6)
(Krishnamurty and Sathyanarayana 1989), 3׳-hydroxyphloretin (7) (Xiao et al. 2017), phlorizin
(8) (Xiao et al. 2017), phloretin-2׳-O-β-d-xyloside (9) (Xiao et al. 2017), phloretin-2׳-O-(β-d-
xylopyranosyl-(1-6)-β-d-glucopyranoside) (10) (Lu and Foo 1997), 5,7,3׳5,׳-tetrahydroxy-di-
hydroflavone (11) (Nessa et al. 2004), (–)-catechin (12) (Kashiwada et al. 1986), (–)-epicatechin
(13) (Lu and Foo 1997), chrysin (14) (Park et al. 2007), quercetin (15) (Sirat et al. 2010), cern-
uoside (16) (Güçlütürk et al. 2012), quercitrin (17) (Sirat et al. 2010), quercetin-3-O-α-L-rham
-
noside (18) (Fukunaga et al. 1988), quercetin-3-O-α-L- arabinofuranoside (19) (Ek et al. 2006),
hyperin (20) (Ek et al. 2006), quercetin-3-O-β-d-xyloside (21) (Ek et al. 2006), and procyanidin
B2 (22) (Khan et al. 1997) by comparison of their spectroscopic data with those reported in
the literature (Figure 1).
Some of the isolated compounds were evaluated for antioxidant activity Table 1.
Compounds 18 and 14 exhibited significant DPPH free radical scavenging activities while

4
Q. HU ET AL.
the compounds 17, 19, 11, 7, 20, 22, 12 and 13 showed moderate activities. The HPLC fin-
gerprint analysis of the fruits showed that they contained mostly the same active compo-
nents as those extracted from the leaves of M. hupehensis (see Supplemental material).
3. Experimental
3.1. General
UV spectra were recorded on a Shimadzu UV-2600 spectrophotometer. IR spectra were
recorded on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr pellets. One-
dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on a Bruker Avance
DRX-600 spectrometer using the residual non-deuterated solvent signal as an internal stand-
ard. ESIMS and HRESIMS were recorded on an Agilent 6520 Q-TOF. HPLC was performed on
a Shimadzu LC-6AD with a Shimadzu C18 (20 × 250 mm) column. Silica gel (100–200 and
200–300 mesh, Qingdao Marine Chemical Co., Ltd., People’s Republic of China) and MCI gel
CHP20/P120 (75–150 μm, Mitsubishi Chemical Corporation, Japan) were used for column
chromatography.
3.2. Plant material
The leaves of M. hupehensis were collected from Shansong Biological Products Co., Ltd. (Linyi,
China), in September 2014. The leaves were identified by Prof. Lan Xiang, and a voucher
specimen (MH201409) has been deposited in the Herbarium of the School of Pharmaceutical
Sciences, Shandong University.
3.3. Extraction and isolation
The aired-dried leaves of M. hupehensis (10 Kg) were extracted with EtOH/H2O (80/20 v/v,
twice, 2 h each time) under reflux conditions to result a crude extract, which was further
subjected to a HPD-100 macroporous adsorption resin chromatography eluted successively
with aqueous EtOH gradient system (0, 30 and 95%). The 30% EtOH fraction (the total
polyphenols) was separated on a polyamide column (EtOH/H2O; 0, 15, 25, 50, 100%) to result
five subfractions, fraction A1-A5. Fraction A1 was separated on a Sephadex LH-20 column
and then on semi-preparative HPLC (15% ACN : H2O) to yield compounds 10 (36 mg), 12
(9 mg), 13 (34 mg), and 22 (24 mg).The main component phlorizin (8; 300 g) was isolated
from fraction A2. Fraction A3 was subjected to silica gel columns then to Sephadex LH-20
columns to yield compounds 7 (20 mg), 16 (80 mg), 17 (16 mg), 18 (60 mg) and 20 (20 mg).
Fraction A4 was subjected to a Sephadex LH-20 column and then semi-preparative HPLC to
yield compounds 19 (10 mg) and 21 (20 mg). Fraction A5 was subjected to a Sephadex LH-20
column to yield compounds 6 (1 g) and 15 (40 mg). The 95% EtOH fraction was suspended
in H2O, respectively, partitioned with petroleum ether, EtOAc, CH2Cl2 and n-BuOH to result
four organic fractions. The CH2Cl2 soluble fraction was subjected to silica gel columns to
yield compound 14 (20 mg).The EtOAc soluble fraction was separated by silica gel columns
and RP-C18 columns to yield compounds 2 (15 mg) and 11 (25 mg). The n-BuOH soluble
fraction was separated by RP-C18 columns and semi-preparative HPLC to isolate compounds
1 (15 mg), 3 (10 mg), 4 (6 mg), 5 (8 mg) and 9 (11 mg).

NATURAL PRODUCT RESEARCH
5
5-O-β- -glucopyranoside-4-chromanone (1):yellow amorphous powder, [α]25 D -55.10
d
(c 0.10, MeOH); UV (MeOH) λmax (log ε):283 (3.22), 321 (2.95) nm; IR (KBr): νmax 3368, 2917,
1
1584, 1403, 1311, 1248, 1194, 1028, 953, 843, 708, 651 cm−1; H NMR (DMSO-d6, 600 MHz)
and 13C NMR (DMSO-d6, 150 MHz) data, see Table S1; positive HRESIMS [M + Na]+: m/z
365.0849 (calcd. for C15H18O9Na, 365.0843).
3.4. Acid hydrolysis of compound 1
Compound 1 (2.0 mg) was dissolved in MeOH (3 mL), then treated with 2 M HCl (1,4-diox-
ane:H2O, 1:1, 2 mL) and heated to 90 °C for 3 h following the literature method (Qin et al.
2016). After the solvent was removed under vacuum, the reaction mixture was extracted
with CHCl3 (5 mL × 3). The aqueous layer was neutralised using 0.5% ammonia solution,
then, evaporated to dryness. The residue was dissolved in anhydrous pyridine (1 mL) followed
by the addition of l-cysteine methyl ester hydrochloride (2 mg) and heated to 60 °C for 1 h.
Then trimethylsilyimidazole solution (1.5 mL) was added and the reaction mixture was
heated to 60 °C for 30 min. The dried product was partitioned with n-hexane and H2O. The
n-hexane layer was analysed by GC. The retention time of d-glucose (tR, 16.95 min) was
confirmed by comparison with an authentic sample.
3.5. DPPH radical scavenging activity
The DPPH assay was performed according to the method described by Huang (Huang et al.
2003).About 100 μL of the sample (concentration ranging from 0.25 to 256 μg/mL) were
added to 100 μL of DPPH solution (150 μM in ethanol). The reaction was carried out at 25 °C
in the dark for 30 min with shaking for 15 s, and then the absorbance of the reaction mixture
was recorded with spectrophotometer at 517 nm. Pure ethanol was used as a control sample.
The radical scavenging activities of the samples, expressed as partial inhibition of DPPH,
were calculated according to the formula: inhibition (%) = [(A0–A1)/A0] × 100, where A0 and
A1 are the absorbance values of the blank sample and of the tested samples, respectively.
Edaravone was used as a positive control. The SC50 (the concentration required to scavenge
50% radicals) values of the tested compounds toward DPPH were calculated using the SPSS
21.0 software.
4. Conclusion
One new 4-chromanone glycoside named as 5-O-β-d-glucopyranoside-4-chromanone (1),
together with 21 known polyphenols, was isolated from the leaves of M. hupehensis.
Antioxidant activity assay showed that compounds 18 and 14 exhibited significant activities
with SC50 values 2.73 and 2.91 μg/mL, respectively. The HPLC fingerprint profiles of the leaves
and fruits extracts exhibited that the constituents were almost the same except for a higher
content of phlorizin present in the leaves.
Disclosure statement
No potential conflict of interest was reported by the authors.

6
Q. HU ET AL.
Funding
This work was financially supported by the National Natural Science Foundation of China [grant num-
ber 81630093].
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